Oligonucleotide N3&#39;→P5&#39; phosphoramidates: hybridization and nuclease resistance properties

ABSTRACT

Modified oligonucleotides 3&#39;-NHP(O)(O - )O-5&#39; phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 08/214,599,filed Mar. 18, 1994.

FIELD OF THE INVENTION

The present invention relates to hybridization and nuclease resistancemethods employing oligonucleotide N3'→P5' phosphoramidates.

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BACKGROUND OF THE INVENTION

Oligonucleotides have been proposed as potent diagnostic compounds andas new rationally designed therapeutic agents (Uhlman, 1990; Helene, etal., 1990; Helene, 1991). The mechanism of action of these compounds isbased on their specific interaction with RNA or DNA regions of interest.

Several modifications of the natural phosphodiester internucleoside bond{phosphomono(Eckstein, et al., 1985; Cohen, 1993) or dithioate(Marshall, et al., 1993), methylphosphonate (Miller, phosphodiesteramidate (Letsinger, et al., Froehler, et al., 1988)} have beenintroduced to improve (i) the stability of the oligomers in biologicalmedia, and (ii) the hybridization properties of the oligomers.

Unfortunately, the vast majority of these analogs exhibit reducedbinding with target RNA or DNA strands via duplex or triplex formation(Kibler-Herzog, et al., 1991). Moreover, the presence of thestereoisomers at phosphorous in some of these analogs may complicate thebinding patterns with complimentary nucleic acids (LaPlauche, et al.,1986; Bower, et al., 1987; Tidd, et al., 1988).

SUMMARY OF THE INVENTION

Methods and compositions of the present invention relate tooligodeoxyribonucleotides having contiguous nucleoside subunits joinedby intersubunit linkages. In the oligonucleotides, at least 2 contiguoussubunits are joined by N3'→P5' phosphoramidate intersubunit, or greaterthan 3 of the total intersubunit linkages are N3'→P5' phosphoramidateintersubunit linkages.

An exemplary N3'→P5' phosphoramidate intersubunit linkage is shown inFIG. 2A, where X is --O, --OR or --R, and R is selected from the groupconsisting of alkyl, alkenyl, aryl, and alkaryl. For definitions ofthese exemplary substituent groups see the Definitions section below.

The nucleoside subunits making up the oligodeoxyribonucleotides of thepresent invention can be selected to be in a defined sequence: such as,a sequence of bases complementary to a single-strand nucleic acid targetsequence or a sequence that will allow formation of a triplex structurebetween the oligodeoxyribonucleotide and a target duplex.

In one embodiment the oligodeoxyribonucleotide has at least 3 contiguoussubunits joined by N3'→P5' phosphoramidate linkages. This grouping oflinkages can, for example, be located at the 3' end of theoligodeoxyribonucleotide. At this location the N3'→P5' phosphoramidatelinkages confer nuclease resistance to the oligodeoxyribonucleotide.

In another embodiment of the present invention, all of the intersubunitlinkages are N3'→P5' phosphoramidate linkages.

Also included in the invention are oligodeoxyribonucleotides where theintersubunit linkages alternate the N3'→P5' phosphoramidate linkage anda second linkage. The second linkage may be selected from one or moredifferent types of linkages, for example, phosphodiester linkages orphosphodiester and phosphorothioate linkages. The second linkage isselected, for example, from the group consisting of phosphodiester,phosphotriester, methylphosphonate, phosphoramidate P3'→N5', andphosphorothioate. In one embodiment at least 50% of the intersubunitlinkages are N3'→P5' phosphoramidate linkages.

The present invention includes a method for generating a triplex DNAmolecule, comprising forming an oligodeoxyribonucleotide as describedabove, where the oligodeoxyribonucleotide has a sequence of nucleosidesubunits effective to form triple-helix structure with a target duplexDNA. The oligodeoxyribonucleotide is then contacted with the duplex DNAunder conditions effective to allow formation of a triplex between theoligodeoxyribonucleotide and the duplex target DNA. This method can becarried out under a variety of conditions, for example, intracellularlyor in solution.

The present invention also includes a triplex DNA molecule, having threeDNA strands: (i) a duplex DNA molecule, having two complementarystrands, and (ii) bound to the duplex a third strandoligodeoxyribonucleotide having N3'→P5' phosphoramidate linkages asdiscussed above. In one embodiment, 50% or greater of the intersubunitlinkages of the third strand oligodeoxyribonucleotide are N3'→P5'phosphoramidate linkages, including fully modifiedoligodeoxyribonucleotides.

Further, the invention includes a method of enhancing the resistance ofan oligodeoxyribonucleotide to nuclease digestion. In this method anoligodeoxyribonucleotide is formed having N3'→P5' phosphoramidatelinkages as described above. The oligodeoxyribonucleotide is exposed tonuclease. Such oligodeoxyribonucleotides are more resistant to nucleasedigestion than a corresponding oligodeoxyribonucleotide having onlyphosphodiester intersubunit linkages. Nuclease resistance is observedintracellularly as well.

The oligodeoxyribonucleotides having N3'→P5' phosphoramidate linkages,as described above, have superior hybridization properties. The presentinvention also includes a method of enhancing hybridization of a firstoligodeoxyribonucleotide to an RNA target sequence, where theoligodeoxyribonucleotide has contiguous nucleoside subunits joined byintersubunit linkages. In the method, a second oligodeoxyribonucleotidehaving N3'→P5' phosphoramidate linkages is formed having the samesequence of contiguous nucleoside subunits as the firstoligodeoxyribonucleotide. The second oligodeoxyribonucleotide iseffective to hybridize to said target RNA sequence. The secondoligodeoxyribonucleotide is then contacted with the RNA under conditionseffective to allow formation of a complex between theoligodeoxyribonucleotide and the RNA. Such contacting can be carried outunder a variety of conditions, including intracellularly.

The present invention also includes a method and kit for the isolationof a target RNA from a sample. The kit includes anoligodeoxyribonucleotide having N3'→P5' phosphoramidate linkages, asdescribed above, where the oligodeoxyribonucleotide is effective tohybridize to the target RNA sequence. Typically, theoligodeoxyribonucleotide is attached to a solid support, such as amagnetic bead, to facilitate isolation.

In another embodiment, the present invention includes a diagnosticmethod to detect the presence in a sample of an RNA having a selectedtarget sequence. In this method, oligodeoxyribonucleotides havingN3'→P5' phosphoramidate linkages are created that are effective to forma hybridization complex with a target sequence. Theoligodeoxyribonucleotide is then contacted with the sample underconditions effective to allow formation of the hybridization complexbetween the oligodeoxyribonucleotide and the target sequence. Thepresence of the hybridization complex is then detected. Detection of thehybridization complex can be accomplished by labelling theoligodeoxyribonucleotide with a reporter moiety, where detectingincludes detection of the reporter moiety. Numerous reporter moletiesare available, including, but not limited to, radioactive labels, biotinlabels, and fluorescent labels. This detection method can be carried outunder a variety of conditions including intracellularly.

Similar diagnostic methods can be carried out usingoligodeoxyribonucleotide having N3'→P5' phosphoramidate linkages, wherethe target sequences are duplex DNA or single-stranded DNA. In the caseof detection of duplex DNA, detection of the hybridization complex canbe accomplished using a gel band shift assay. For detection ofsingle-stranded DNA the oligodeoxyribonucleotide typically containsgreater than 50% of the total intersubunit linkages as N3'→P5'phosphoramidate intersubunit linkages.

The present invention also includes a duplex oligodeoxyribonucleotide,having (i) two complementary strands, and (ii) contiguous nucleosidesubunits joined by intersubunit linkages, where at least 2 contiguoussubunits are joined by N3'→P5' phosphoramidate intersubunit linkages, orgreater than 3 of the total intersubunit linkages are N3'→P5'phosphoramidate intersubunit linkages (as described above). In oneembodiment, 50% or greater of the intersubunit linkages of at least onestrand are N3'→P5' phosphoramidate linkages. In another embodiment allof the intersubunit linkages of at least one strand are N3'→P5'phosphoramidate linkages. Such duplex DNA molecules may also include aflexible hinge region connecting the complementary strands. The hingeregion may connect the strands in any desired polarity, e.g., 5' to 3',3' to 5', 3' to 3', and 5' to 5'.

Further, the present invention includes a method of forming a triplexnucleic acid complex, having two complementary DNA strands and one RNAstrand containing a target region, and compositions thereof. In themethod, an oligodeoxyribonucleotide having contiguous nucleosidesubunits Joined by intersubunit linkages is formed. Theoligodeoxyribonucleotide is capable of forming a duplexoligodeoxyribonucleotide, having (i) two complementary strands with 5'and 3' ends, (ii) contiguous nucleoside subunits joined by intersubunitlinkages, where at least 2 contiguous subunits are joined by N3'→P5'phosphoramidate intersubunit, or greater than 3 of the totalintersubunit linkages are N3'→P5' phosphoramidate intersubunit linkages(as described above), (iii) where the strands are connected from the endof one strand to the end of the other strand by a flexible hinge region,and (iv) the complementary oligodeoxyribonucleotide strands having asequence of nucleoside subunits effective to form triple-helix structurewith the RNA target. The oligodeoxyribonucleotide is then contacted withthe RNA target under conditions effective to allow formation of atriplex between the oligodeoxyribonucleotide and the RNA. This methodcan be carried out under a variety of conditions, including,intracellularly.

The present invention also includes pharmaceutical compositions ofoligodeoxyribonucleotides having N3'→P5' phosphoramidate linkages, asdescribed above. The oligodeoxyribonucleotides are useful in therapeuticapplications based on hybridization, such as, antigene and antisenseapplications.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1D show the structures of subunits useful for the synthesisof oligonucleotides having internucleoside 3'-NHP(O)(O⁻)O-5'phosphoramidate linkages (N3'→P5'). FIG. 1E shows a schematic outline ofthe step-by-step synthesis of uniformly modified oligonucleotides. Inthe figure, CE=cyanoethyl and CPG=controlled pore glass.

FIGS. 2A to 2E present exemplary combinations of 3'-NHP(O)(O⁻)O-5'phosphoramidate intersubunit linkages with other, alternative linkages.In FIGS. 2D and 2F, R' is, for example, a lower alkyl group, othersubstitutions are possible as described by Goodchild (1990).

FIG. 3 presents exemplary oligonucleotides and T_(m) values of duplexesand triplexes. In the figure:

¹ =The T_(m) 's of complexes in the buffer A.

^(b) =T_(m) in the buffer; T_(m) of the hairpin duplex was 55.7° and61.5° C. in buffer A and B, respectively.

^(c) =Mismatched nucleotide is underlined.

FIGS. 4A to 4C. FIG. 4A presents an IE HPLC profile of the reactionmixture after synthesis of the phosphoramidate Oligonucleotide SEQ IDNO:3. FIG. 4B presents a capillary gel electrophoresis profile of thereaction mixture after synthesis of the undecaphosphoramidate SEQ ID NO:6. FIG. 4C shows the results of ³¹ P-NMR of the decaphosphoramidate 3.

FIGS. 5A to 5B display melting curves for the duplexes, formed byphosphodiester and phosphoramidate oligomers.

FIG. 6 shows exemplary oligonucleotide hairpins and their T_(m) values.

FIGS. 7A to 7D show melting curves for the triplexes.

FIG. 8 shows gel-electrophoresis analysis of the oligonucleotide triplexformation under native conditions.

FIG. 9 shows gel-electrophoresis analysis of the oligonucleotide triplexformation under native conditions.

FIGS. 10 through 19 show the results of anti-sense oligonucleotides,having either phosphoramidate (N3'→P5') (FIGS. 10-15) orphosphorothioate (FIGS. 16-19) intersubunit linkages, on leukemia cellproliferation for different BCR-ABL leukemia cell lines and control celllines.

FIG. 20 schematically represents the preparation of 3'-amino-N⁶-benzoyl-5'-dimethoxytrityl-2',3'-dideoxyadenosine.

DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

An "alkyl group" refers to an alkyl or substituted alkyl group having 1to 20 carbon atoms, such as methyl, ethyl, propyl, and the like. Loweralkyl typically refers to C₁ to C₅. Intermediate alkyl typically refersto C₆ to C₁₀. Similarly, "cycloalkyl group" refers to a saturatedcarbocyclic ring group which may have alkyl, aryl, aralkyl substituentssuch as cyclopropyl, cyclopentyl, cyclohexyl, and the like, or asubstituted form thereof.

An "alkenyl group" refers to a hydrocarbon group containing acarbon-carbon double bond, such as vinyl, allyl, cyclopentenyl, and thelike. An "alkenyl group" also refers to substituted alkenyls.

An "aryl group" refers to an aromatic ring group having 5-20 carbonatoms, such as phenyl, naphthyl, anthryl, or substituted aryl groups,such as, alkyl- or aryl-substitutions like tolyl, ethylphenyl,biphenylyl, etc. Also included are heterocyclic aromatic ring groupshaving one or more nitrogen, oxygen, or sulfur atoms in the ring.

An "alkaryl group" refers to substituted alkyl group, such as,aryl-substitutions like benzyl, phenethyl, etc.

By "substituted" it is generally meant that the group is derivatizedwith one or more small chemical moieties,.e.g., methoxy, ethoxy,halogen, hydroxyl, cyano, amido, amine and ethylene oxide. Any of thegroups defined above may be substituted, for example, (--CF₃).

"Oligonucleotides" typically refer to nucleoside subunit polymers havingbetween about 4 and about 50 contiguous subunits. The nucleosidesubunits can be joined by a variety of intersubunit linkages, including,but not limited to, those shown in FIGS. 2A to 2E. Further,"oligonucleotides" includes modifications, known to one skilled in theart, to the sugar backbone (e.g., ribose or deoxyribose subunits), thesugar (e.g., 2' substitutions), the base, and the 3' and 5' termini."Oligodeoxyribonucleotides" include such modifications, such as, 2'sugar substitutions of flourine.

"Nucleoside" is defined herein as a pentose sugar (a ribose,deoxyribose, or modification thereof) bound to a base capable forminghydrogen bonds (typically a purine or pyrimidine).

A "base" is defined herein to include (i) typical DNA and RNA bases(uracil, thymine, adenine, guanine, and cytosine), and (ii) modifiedbases or base analogs (e.g., 5-methyl-cytosine, 5-bromouracil, orinosine). base analog is a chemical whose molecular structure mimicsthat of a typical DNA or RNA base.

II. THE PRESENT INVENTION

Experiments performed in support of the present invention demonstratethat oligonucleotides containing achiral internucleoside3'-NHP(O)(O⁻)O-5' phosphoramidate linkages (N3'→P5') are more resistantto nuclease digestion and have improved RNA and dsDNA hybridizationcharacteristics relative to oligonucleotides not containing N3'→P5'phosphoramidate linkages. Oligonucleotides containing the N3'→P5'linkages have excellent antisense activity against complementary mRNAtargets in in vitro cell growth inhibition assays. Further, theoligonucleotides exhibit low cytotoxicity.

A. SYNTHESIS AND CHARACTERIZATION OF OLIGONUCLEOTIDES CONTAININGINTERNUCLEOSIDE 3'-NHP(O)(O⁻)O-5' PHOSPHORAMIDATE LINKAGES

Oligonucleotides, containing single N3'→P5' phosphoramidate linkage wereprepared by chemical ligation in aqueous media (essentially as describedby Shabarova, 1988). Alternatively, oligonucleotides containing N3'→P5'linkages between two subunits where the next intersubunit bond was atleast one phosphodiester bond, were synthesized on a solid support viacoupling of the preformed phosphoramidate dimer blocks (Gryaznov, etal., 1992; Mag, et al., 1992). Random size ribooligonucleotide N3'→P5'phosphoramidates were obtained via self-polymerization of dimer blocks(Zielinski, et al., 1987). Azhayer, et al., describe the synthesis ofdefined sequence oligoribonucleotides.

The present invention includes solid support synthesis methods for thegeneration of oligodeoxyribonucleotides with contiguous nucleosidesubunits joined by N3'→P5' phosphoramidate intersubunit linkages (np)(Example 1, FIG. 1). Sequential synthesis of oligodeoxyribonucleotidesutilizes 5'-dimethoxytrityl-3'-amino-deoxyribonucleotide subunits. Thepreparation of each of these subunits is described in Example 1 (see,for example, FIG. 20).

Oligonucleotides having contiguous subunits joined by N3'→P5'phosphoramidate intersubunit linkages (e.g., uniformly modified) weresynthesized on a solid support using the step-by-step elongationprocedure outlined in FIG. 1 (Example 1). The synthetic cycle foraddition of a single aminonucleoside consists essentially of thefollowing operations: detritylation (FIG. 1, step i); phosphitylation ofthe 5'- hydroxyl group to generate a polymer supported 5'-H-phosphonatediester (FIG. 1, steps ii, iii); Atherton-Todd type (Atherton, et al.,1945; Gryaznov, et al., 1992; Gryaznov, et al., 1986; Gryaznov, et al.,1990) coupling of a 5'-dimethoxytrityl-3'-amino nucleoside (Glinski, etal., 1970) with the 5'-H-phosphonate in the presence of carbontetrachloride (FIG. 1, step iv). This cycle can be repeated severaltimes resulting in phosphoramidate oligonucleotide after deprotectionwith ammonia (FIG. 1, step v, vi). Average coupling yields were 94%-96%per step as judged by dimethoxytrityl (DMT) cation assay. Exemplaryoligodeoxyribonucleotides containing N3'→P5' phosphoramidate linkages("np") are presented in FIG. 3.

Oligodeoxyribonucleotides of the present invention contain at least twocontiguous subunits joined by N3'→P5' phosphoramidate intersubunitlinkages (for example, 5'-T-np-G-np-A-3'), or greater than 3 totalN3'→P5' phosphoramidate intersubunit linkages. In one embodiment, theoligodeoxyribonucleotides contain fully modified N3'→P5' phosphoramidateintersubunit linkages (e.g., FIG. 3, experiment 13, oligonucleotide SEQID NO:6). In another embodiment, the oligodeoxyribonucleotides havealternating N3'→P5' phosphoramidate intersubunit linkages betweensubunits, typically alternating with phosphodiester or phosphorothioatelinkages (see exemplary linkages below and in FIG. 2). A example of suchan oligodeoxyribonucleotide having alternating linkages is shown in FIG.3, experiment 3, oligonucleotide SEQ ID NO:2. Synthesis ofoligonucleotide SEQ ID NO:2 is described in Example 1.

Oligonucleotides were isolated by ion exchange high performance liquidchromatography (IE HPLC; Example 2, FIG. 4A). Purities of isolatedoligonucleotide preparations were evaluated by capillary electrophoresisand slab gel electrophoresis analysis (Example 2, FIG. 4B).

Presence of the phosphoramidate linkages in the purifiedoligonucleotides was confirmed by ³¹ P-NMR (Example 2, FIG. 4C) and byselective acid-catalyzed hydrolysis of phosphoramidate linkages (Example2).

The cyanoester group in FIG. 1E (step i, iii) can be replaced by otherpendent groups, including, alkyl (usually lower or intermediate alkyls),alkenyl, aryl and alkaryl groups (or substitutions of any of thepreceding groups). Typically, such pendant groups do not interfere withthe synthesis of oligonucleotides or the ability of the oligonucleotideto hybridize to a target. One exemplary pendent group is --C₃ (Gryaznov,et al., 1992). A typical repeat unit is shown in FIG. 2A, where "X" is"--O⁻ ", "--OR" or "--R", and "R" is, for example, any of the followingpendent groups or substitutions thereof: alkyl, alkenyl, aryl, andalkaryl.

In addition to phosphoramidate analogs and chimeticphosphoramidate/phosphodiester analogs (FIG. 2C), the internucleosideN3'→P5' phosphoramidate linkages can be incorporated intooligonucleotides having one or more other modified intersubunit linkages(reviewed by Goodchild, 1990), including, but not limited to,phosphotriesters (FIG. 2D), methylphosphonates (FIG. 2B),phosphoramidates (FIG. 2F), phosphoramidates P3'→N5', andphosphorothioates (FIG. 2E).

B. NUCLEASE RESISTANCE OF OLIGONUCLEOTIDES CONTAINING ACHIRALINTERNUCLEOSIDE 3'-NHP(O)(O⁻)O-5' PHOSPHORAMIDATE LINKAGES

Stability of the oligonucleotide phosphoramidates toward hydrolysis bysnake venom phosphodiesterase was evaluated in comparison with naturalphosphodiester compounds (see Materials and Methods). Phosphodiesterdecamer Oligonucleotide SEQ ID NO:1 (FIG. 3) was treated with snakevenom phosphodiesterase. The oligonucleotide SEQ ID NO:1 was completelyhydrolyzed after 10 minutes, as Judged by reversed phase highperformance liquid chromatography HPLC.

In contrast, phosphoramidate analog Oligonucleotide SEQ ID NO:3 wasessentially intact even after 50 minutes of treatment with snake venomphosphodiesterase. After 4.5 hours, approximately 50% of OligonucleotideSEQ ID NO:3 was converted to the presumed 9-mer (TnpT)₄ T_(NH2) with aterminal 3'-amino group. The presence of the terminal 3'-amino groupretarded further digestion of the oligomer. After 22 hours ofhydrolysis, the starting 10-mer oligonucleotide SEQ ID NO:3 wascompletely converted to the 3'-amino-terminal 9-mer {(TnpT)₄ T_(NH2) }.Only about 20% further digestion of the {(TnpT)₄ T_(NH2) } compound wasobserved.

These results demonstrate the increased nuclease resistance ofoligonucleotides containing N3'→P5' phosphoramidate linkages ("np"),relative to oligonucleotides having standard phosphodiester backbones.In one embodiment of the present invention, nuclease resistance ofoligodeoxyribonucleotides is generated by placing approximately 3contiguous subunits linked by N3'→P5' phosphoramidate intersubunitlinkages at the 3' end of the oligodeoxyribonucleotides.

C. HYBRIDIZATION PROPERTIES OF OLIGONUCLEOTIDES CONTAINING N3'→P5'PHOSPHORAMIDATE LINKAGES

The hybridization properties of the phosphoramidate analogs wereevaluated relative to complementary DNA or RNA strands having standardphosphodiester intersubunit linkages. The thermal stability data forduplexes generated from phosphoramidate analogs and phosphodiesteroligomers are summarized in FIG. 3 (Example 1).

Exemplary melting curve data (Example 4A) for duplexes formed byphosphodiester and phosphoramidate analog oligomers are presented inFIGS. 5A and 5B. In the figures, curves (A), (C), (B), and (D)correspond to experiments 8, 9, 13 and 14 in FIG. 3, respectively.

Substitution of the internucleoside phosphodiester for the N3'→P5'phosphoramidate linkages dramatically changed the oligonucleotides'hybridization properties. Melting temperatures (T_(m) 's) of duplexesformed by the entirely modified 10-mer Oligonucleotide SEQ ID NO:3 withpoly Da (i.e., DNA) and poly A (i.e., RNA) were 36.0° C. and 51.5° C.,respectively (FIG. 3, experiments 5 and 6). These Tm's are 6.3° C. and24.5° C. higher than duplexes formed by the phosphodiester counterpartOligonucleotide SEQ ID NO:1 with poly Da and poly A (FIG. 3, experiments1 and 2).

The same trend is true for the mixed-base undecanucleotide SEQ ID NO:6(FIG. 3), where the T_(m) 's of duplexes with complementary DNA and RNAstrands were 49.2° C. and 72.4° C., respectively, (FIG. 3, experiments13, 14). These values are 11.7° C. and 22.9° C. higher than for theparent phosphodiester compound Oligodeoxyribonucleotide SEQ ID NO:4(FIG. 3, experiments 8 and 9). Also, the duplex with the same RNA targetformed by phosphoramidate 11-mer Oligodeoxyribonucleotide SEQ ID NO:6 ismore stable (by 18.0° C.) than one formed by the homologous RNA oligomerSEQ ID NO:5 (FIG. 3, experiment 11).

Oligodeoxyribonucleotide SEQ ID NO:2, with alternatingphosphodiester--phosphoramidate linkages, also binds more tightly withthe RNA strand, T_(m) 33.7° C. (FIG. 3, experiment 4) than thecorresponding phosphodiester compound (Oligodeoxyribonucleotide SEQ IDNO:1, FIG. 3, experiment 2). However, Oligodeoxyribonucleotide 2 bindsless strongly with the DNA template, T_(m) 25.8° C., (FIG. 3, experiment3) relative to its phosphodiester counterpart (OligodeoxyribonucleotideSEQ ID NO:1, FIG. 3, experiment 1).

Hybridization of the phosphoramidate oligonucleotides with complementarynucleic acids is sequence specific and determined by the properWatson-Crick base pairing. The duplex formed by phosphoramidateOligodeoxyribonucleotide SEQ ID NO:6 with single mismatched RNA target(FIG. 3, experiment 15) is substantially less stable (ΔT_(m) -12.2° C.)than the duplex formed with the fully complementary RNA oligomer (FIG.3, experiment 13). About the same mismatch discrimination was observedfor the phosphodiester deoxyribo- and ribo- oligonucleotides, whereΔT_(m) was -14.4° C. and -12.4° C. respectively (FIG. 3, experiments 10,12).

A previous study with phosphoramidate analogs demonstrated thatintroduction of three N3'→P5' phosphoramidate linkages resulted in adestabilization trend, relative to two such linkages, for heteroduplexesformed with deoxyribooliognucleotide targets (Gryaznov, et al., 1992).In contrast to the prior art trend, the results presented abovedemonstrate that typically having up to 50% of the intersubunit linkagesas phosphoramidate linkages decreases the stability of DNA/DNAheteroduplexes. Greater than 50% phosphoramidate intersubunit linkagesin one strand of a DNA duplex, however, begins to improve stability ofthe duplex relative.

When a DNA duplex is formed between a normal, phosphodiesteroligonucleotide and an oligonucleotide fully modified with N3'-P5'phosphoramidate linkages, the thermal stability of the duplex is muchhigher than the corresponding duplex having only phosphodiester linkagesin both strands (FIG. 3, compare experiments 1 and 5).

Gryaznov, et al. (1992) only contains data concerning the hybridizationproperties of DNA/DNA duplexes where one strand contains up to threeN3'→P5' phosphoramidate linkages (non-contiguous). In sharp contrast tothe teachings of the prior art concerning DNA targets, experimentsperformed in support of the present invention demonstrate that havingincreasing numbers of phosphoramidate analog linkages present in anoligodeoxyribonucleotide increases the stability of DNA/RNAheteroduplexes. To achieve DNA/RNA heteroduplex stabilization, apreferred embodiment of the present invention includes anoligodeoxyribonucleotide having at least 2 contiguous, or more than 3total intersubunit linkages modified to have N3'→P5' phosphoramidatelinkages.

Experiments were also performed to evaluate the stability of duplexesformed by oligonucleotides containing phosphoramidate linkages in bothcomplementary strands. Several chimeric phosphoramidate-phosphodiesterhairpin oligomers (FIG. 6) were synthesized (FIG. 6, Example 4B) havingThymidine-containing hinge regions (T₄, FIG. 6). Melting curves obtainedfor these compounds show that the most stable duplexes were formed bythe hairpins in Oligonucleotides SEQ ID NO:9 and SEQ ID NO:2--where bothstrands contain phosphoramidate linkages in opposing positions (FIG. 6,experiments 3, 6).

Also, duplexes formed from single-strand DNA molecules (i.e., hairpins)where one strand contains alternating phosphoramidate-phosphodiesterlinkages and the complementary strands has only phosphodiester linkages,are more stable than their solely phosphodiester counterparts (FIG. 6,experiments 1, 4).

These results suggest that when both strands of a DNA/DNA duplex containphosphoramidate analog linkages, the duplex is stabilized by thepresence of N3'→P5' phosphoramidate linkages in each strand. Stableduplexes can be formed with one phosphoramidate linkage in eachstrand--in one embodiment the phosphoramidate linkage is in the samelocation in each strand.

To achieve DNA/DNA duplex stabilization typically 2 or more of theintersubunit linkages in each DNA strand of a duplex formingoligonucleotide are modified to have N3'→P5' phosphoramidate linkages.In one embodiment, one strand of a hairpin forming DNA oligonucleotidecan be modified to have about 50%-100% of N3'→P5' phosphoramidateintersubunit linkages.

D. TRIPLEX FORMATION USING PHOSPHORAMIDATE ANALOGS

The ability of the phosphoramidate analogs to form triplexes withdouble-stranded DNA was also evaluated (Example 4). Melting curves wereobtained for triplexes formed by the decathymidilic phosphoramidateOligodeoxyribonucleotide SEQ ID NO:6 and the dA₁₀ :dT₁₀ duplex region ofthe hairpin DNA target d(A₁₀ C₄ T₁₀) (FIG. 3, experiment 7). The triplexhad a T_(m) of 32° C. at close to physiological conditions. A morestable triplex (T_(m) 42.2° C.) was observed in magnesium-containingbuffer (FIG. 3, experiment 7).

The same T_(m) value was obtained for triplexes formed byphosphoramidate Oligonucleotide SEQ ID NO:3 with poly Da:poly Dt duplex.Thermal dissociation of the triplexes was monitored by change ofabsorbance at 260 nm (FIGS. 7A and 7C), as well as at 284 nm (FIGS. 7Band 7D), which is characteristic for T:AT triplexes (Riley, et al.,1966).

Results of the gel-shift experiments under native conditions alsodemonstrate formation of the stable triplex by phosphoramidate decamerSEQ ID NO:3 and dsdna target (FIG. 8), as well as stable triplexformation by Oligonucleotide SEQ ID NO:6 (FIG. 9).

Under the same hybridization conditions neither phosphodiesterdecathymidilic acid Oligonucleotide SEQ ID NO:1, nor Oligonucleotide SEQID NO:4 formed triplexes with the same double-stranded DNA targets, asjudged by the melting curves and by the gel-shift experiments (FIG. 8).That is, no corresponding triplex was formed by the oligonucleotideswhich have phosphodiester intersubunit linkages--suggesting that thephosphoramidate analogs may more readily form triplexes than theirphosphodiester containing counterparts.

Similar results were obtained when Oligonucleotide SEQ ID NO:6 andOligonucleotide SEQ ID NO:1 were evaluated for their ability to formtriplex structures with duplex DNA targets (Example 5, FIG. 9).

The results presented above suggest that the phosphoramidateoligonucleotides are more effective for triplex formation, with a duplexsubstrate, than standard phosphodiester oligonucleotides.

III. APPLICATIONS OF OLIGONUCLEOTIDES CONTAINING INTERNUCLEOSIDE3'-NHP(O)(O⁻)O-5' PHOSPHORAMIDATE LINKAGES

Oligonucleotide 3' -NHP (O)(O⁻)O-5' phosphoramidates were synthesized.These compounds are nuclease resistant and form surprisingly stablecomplexes with ssRNA and DNA targets. The N3'→P5' phosphoramidateanalogs have great potential for anti-sense and anti-genediagnostic/therapeutic applications. In a preferred embodiment of thepresent invention, the oligonucleotides are oligodeoxyribonucleotides.

A. ANTI-SENSE APPLICATIONS

Antisense therapy involves the administration of exogenousoligonucleotides that bind to a target nucleic acid, typically an RNAmolecule, located within cells. The term antisense is so given becausethe oligonucleotides are typically complementary to mRNA molecules("sense strands") which encode a cellular product.

The phosphoramidate analog oligonucleotides described herein are usefulfor antisense inhibition of gens expression (Matsukura et al., 1989;Agrawal et al., 1989; Zamecnik et al., 1986; Rittner and Sczakiel, 1991;Stein and Cheng, 1993). Oligonucleotides containing N3'→P5'phosphoramidate linkages have therapeutic applications for a largenumber of medically significant targets, including, but not limited toinhibition of cancer cell proliferation and interference with infectiousviruses. The N3'→P5' phosphoramidate oligonucleotides are useful forboth veterinary and human applications. The low cytotoxicity of thesecompounds and their ability to act effectively as antisense molecules atlow concentrations (see below) make these oligonucleotides highlydesirable as therapeutic antisense agents.

Anti-sense agents typically need to continuously bind all target RNAmolecules so as to inactivate them or alternatively provide a substratefor endogenous ribonuclease H (Rnase H) activity. Sensitivity ofRNA/oligonucleotide complexes, generated by the methods of the presentinvention, to Rnase H digestion can be evaluated by standard methods(Donia, et al., 1993; Kawasaki, et al., 1993).

The methods of the present invention provide several advantages over themore conventional anti-sense agents. First, phosphoramidate analogoligonucleotides bind more strongly to RNA targets than correspondingphosphodiester oligonucleotides. Second, the phosphoramidate analogoligonucleotides are more resistant to degradation by cellularnucleases. Third, in cellular uptake of the compound, an unchargedphosphoramidate analog backbone may allow more efficient entry of thephosphoramidate analog oligonucleotides into cells than a chargedoligonucleotide.

Further, when an RNA is coded by a mostly purine strand of a duplextarget sequence, phosphoramidate analog oligonucleotides targeted to theduplex also have potential for inactivating the DNA--i.e., the abilityto inactivate a pathogen in both single-stranded and double-strandedforms (see discussion of anti-gene therapies below).

Sequence-specific phosphoramidate analog binding molecules arepotentially powerful therapeutics for essentially any disease orcondition that in some way involves RNA. Exemplary modes by which suchsequences can be targeted for therapeutic applications include:

a) targeting RNA sequences expressing products involved in thepropagation and/or maintenance infectious agents, such as, bacteria,viruses, yeast and other fungi, for example, a specific mRNA encoded byan infectious agent;

b) formation of a duplex molecule that results in inducing the cleavageof the RNA (e.g., Rnase H cleavage of RNA/DNA hybrid duplex molecules);

c) blocking the interaction of a protein with an RNA sequence (e.g., theinteraction of TAT and TAR, see below); and

d) targeting sequences causing inappropriate expression or proliferationof cellular genes: for example, genes associated with cell cycleregulation; inflammatory processes; smooth muscle cell (SMC)proliferation, migration and matrix formation (Liu, et al., 1989);certain genetic disorders; and cancers (protooncogenes). In oneembodiment, translation or RNA processing of inappropriately expressedcellular genes is blocked.

Exemplary potential target sequences are protooncogenes, for example,including but not limited to the following: c-myc, c-myb, c-fos, c-kit,ras, and BCR/ABL (e.g., Wickstrom; Zalewski, et al., 1993; Calabretta,et al., 1992, 1993;), oncogenes/tumor suppressor genes (e.g., p53,Bayever, et al.), transcription factors (e.g., NFκB, Cogswell, et al.,1993) and vital genes (e.g., papillomaviruses, Cowsert, et al.; herpessimplex virus, Kulka, et al.). To further illustrate, two RNA regions ofthe HIV-1 protein that can be targeted by the methods of the presentinvention are the REV-protein response element (RRE) and the TAT-proteintransactivation response element (TAR). REV activity requires thepresence of the REV response element (RRE; SEQ ID NO:23), located in theHIV envelope gene (Malim et al., 1989a, 1989b).

The RRE has been mapped to a 234-nucleotide region thought to form fourstem-loop structures and one branched stem-loop structure (Malim et al.,1989a). Data obtained from footprinting studies (Holland et al., 1990;Kjems et al., 1991) suggest that REV binds to six base pairs in one stemstructure and to three nucleotides in an adjacent stem-loop structure ofthe RRE. A minimum REV binding region of about 40 nucleotides instem-loop II has been identified by Cook, et al. (1991; SEQ ID NO:24).This binding region can be target for generation of RNA/DNA duplexes(e.g., Li, et al., 1993) using one or more oligonucleotides, accordingto the methods of the present invention.

The HIV-1 TAT is essential for vital replication and is a potenttransactivator of long terminal repeat (LTR)-directed viral geneexpression (Dayton et al., 1986; Fisher et al., 1986). Transactivationinduced by TAT protein requires the presence of the TAR element (SEQ IDNO:25) which is located in the untranslated 5' end of the viral mRNAelement. The TAR element is capable of forming a stable stem-loopstructure (Muesing et al., 1987). The integrity of the stem and a 3nucleotide (nt) bulge on the stem of TAR has been demonstrated to beessential for specific and high-affinity binding of the TAT protein tothe TAR element (Roy et al., 1990; Cordingley et al., 1990; Dingwall etal., 1989; Weeks et al., 1990). This region can be targeted foranti-sense therapy following the method of the present invention.

In addition to targeting the RNA binding sites of the REV, RRE and TATproteins, the RNA coding sequences for the REV and TAT proteinsthemselves can be targeted in order to block expression of the proteins.

Initial screening of N3'→P5' phosphoramidate oligonucleotides, directedto bind potential antisense target sites, typically includes testing forthe thermal stability of resultant RNA/DNA duplexes. When aphosphoramidate analog oligonucleotide is identified that binds aselected RNA target sequence, the analog is further tested forinhibition of RNA function in vitro. Cell culture assays systems areused for such in vitro analysis (e.g., herpes simplex virus, Kulka, etal.; HIV-1, Li, et al., Vickers, et al.; coronary smooth muscle cellproliferation in restenosis, Zalewski, et al.; IL-2R, Grigoriev, et al.;c-myb, Baer, et al.; c-fos, Cutry, et al.; BCR/ABL, Szczylik, et al.,1991).

Example 5 presents the results of testing phosphoramidateoligonucleotides in one such cell culture system. The assay measuresselective inhibition of leukemia cell proliferation by BCR-ABL antisenseoligonucleotides (Szczylik, et al., 1991). BCR-ABL transcripts are foundin the majority of chronic myelogenous leukemia (CML) patients and inPh⁺ acute lymphocytic leukemia patients, and are believed to benecessary for the maintenance of leukemic phenotype (Gale, et al.;Collins, et al., 1984; Daley, et al.). The BCR-ABL transcripts are theresult of a translocation of the proto-oncogene ABL (chromosome 9) tothe breakpoint cluster region (BCR) (chromosome 22), resulting in theformation of BCR-ABL hybrid genes.

A fully modified N3'→P5' phosphoramidate anti-sense oligonucleotide11-mer (SEQ ID NO:6), complementary to the identified BCR-ABL JunctionB2A2 (cell line BV173), was synthesized and purified. A correspondingoligonucleotide 16-mer (SEQ ID NO:26), (i) containing the 11-mersequence as given above, and (ii) having fully modifies phosphorothicateintersubunit linkages, was also prepared. The oligonucleotides wereadministered to the cells at 24 hour intervals for three days (days 0, 1and 2) at the concentrations shown in FIGS. 10 to 15, 16 and 18.Concentrations in the figures are presented as follows: 40/20/20corresponds to concentrations, in μg/ml, of oligonucleotides as added tothe cell cultures (Example 5).

FIGS. 17 and 18 present the results for experiments performed using a16-mer, having fully modified phosphorothioate intersubunit linkages,with sequence mismatches to the BV173 BCR/ABL splice junction.

The results presented in FIGS. 10, 12 and 14 demonstrate that thephosphoramidate oligonucleotides were extremely effective at inhibitingBV173 leukemia cell proliferation, regardless of the concentration atwhich the oligonucleotide was administered.

The results presented in FIGS. 11, 13 and 15 demonstrate that thephosphoramidate oligonucleotides have negligible cytotoxicity. In theseexperiments, the cell line was either HL60, which contains no BCR/ABLbreakpoint, or K562, which contains the B3A2 BCR/ABL break point, whichis partly non-homologous to the B2A2 BCR/ABL break point.

On the other hand, when similar experiments were performed using thephosphorothioate 16-mer, the oligonucleotides were not effective atinhibiting BV173 leukemia cell proliferation when administered atcomparable concentrations to the phosphoramidate oligonucleotide (FIGS.16 and 18). Specifically, leukemia cells appear to begin to be releasedfrom inhibition by the phosphorothioate 16-mer at a concentration ofabout 1.25/0,625/0.625 compared to a similar release of inhibition seenwhen using the phosphoramidate oligonucleotide at 0.0196/0.0098/0.0098.Accordingly, the results demonstrate that the phosphoramidateoligonucleotides are effective antisense agents at considerably lowerconcentrations than the widely used phosphorothioate oligonucleotides.

Further, the inhibition curves seen with the phosphoramidateoligonucleotide treatment have a downward inflection at later timepoints, even after the partial release of inhibition (FIG. 14). On theother hand, the inhibition curves seen with the phosphorothioateoligonucleotide treatment have a steep upward inflection at later timepoints, after the partial release of inhibition (FIG. 18). These resultssuggest that the release from inhibition is more dramatic for thephosphorothioate oligonucleotides when compared to the phosphoramidateoligonucleotides.

The absence of cytotoxicity with the phosphorothioate 16-mer wascomparable to that seen with the phosphoramidate oligonucleotide appliedat the same concentration.

The results presented above confirm in cell culture the superiorqualities of the N3'→P5' phosphoramidate oligonucleotides demonstratedby the hybridization studies described above. These results support theusefulness and efficacy of the N3'→P5' phosphoramidate oligonucleotidesin antisense and antigene in vivo therapies.

Further, the results demonstrate that the N3'→P5' phosphoramidateoligonucleotides provide superior antisense function in vitro thanphosphorothioate oligonucleotides. To date, the phosphorothioatebackbone modification in oligonucleotides has become the standard forantisense applications, representing the subject analog in more than 95%of the ˜2500 antisense journal publications in 1993.

In a great variety of assay systems involving a large number of mRNAtargets, the antisense phosphorothioate oligonucleotides were requiredat concentrations of 1-15 μM in order to achieve substantial inhibitoryeffects. Nevertheless, inhibitory activity at that level was sufficientto commence US FDA approved clinical trials in three diseases targetingthree different mRNAs: CML (chronic myelogenous leukemia), IND #42974and AML (acute myelogenous leukemia) IND #40453 (Antiviral AgentsBulletin, Vol. 5, No. 6, pp161-162 (1992), ISSN 0897-9871, BiotechnologyInformation Institute); as well as Hepatitis B Virus (HBV).

The data presented in Example 5 demonstrate that the N3'→P5'phosphoramidate oligonucleotides are effective antisense agents at muchlower concentrations than corresponding phosphorothioates. Further, theN3'→P5' phosphoramidate oligonucleotides have no apparent cytotoxicityeven at the highest concentrations used, which were well above theconcentrations required for antisense activity. The results indicatethat the N3'→P5' phosphoramidate oligonucleotides are excellent agentsfor therapeutic applications.

In vitro effects of a selected phosphoramidate analog oligonucleotidecan be confirmed in an in vivo system. Such in vivo systems include, butare not limited to, the following (target--model system): hepatitisvirus--chimpanzee or monkey models; c-myb, c-myc, bcr-abl--SCID mousemodels (e.g., Ratajczak, et al.); NF-κB--mouse (Higgins, et al.); andp120-mouse (Perlakey, et al.).

B. ANTI-GENE APPLICATIONS

Inhibition of gens expression via triplex formation has been previouslydemonstrated (Coohey et al., 1989; Orson et al., 1991; Postel et al.,1991). The increased stability of triplex structures formed whenemploying third strand phosphoramidate analog oligonucleotides providesa stronger tool for antigens applications, including veterinary andhuman therapeutic applications.

A target region of choice is selected based on known sequences usingstandard rules for triplex formation (Helene and Toulme, 1990).Typically, the phosphoramidate analog nucleic acid sequence is targetedagainst double-stranded genetic sequences in which one strand containspredominantly purines and the other strand contains predominantlypyrimidines.

Phosphoramidate analog oligonucleotides of the present invention aretested for triplex formation against a selected duplex target sequencesusing band shift assays (Example 4). Typically, high percentagepolyacrylamide gels are used for band-shift analysis and the levels ofdenaturing conditions (Ausubel et al.; Sauer et al.; Sambrook et al.)are adjusted to reduce any non-specific background binding.

The duplex target is labeled (for example, using a radioactivenucleotide) and mixed with a third strand oligonucleotide, being testedfor its ability to form triplex structures with the target duplex. Ashift of the mobility of the labelled duplex oligonucleotide indicatesthe ability of the oligonucleotide to form triplex structures.

Triplex formation is indicated in the band shift assay by a decreasedmobility in the gel of the labeled triplex structure relative to thelabeled duplex structure.

Numerous potential target sites can be evaluated by this methodincluding target sites selected from a full range of DNA sequences thatvary in length as well as complexity. Sequence-specific phosphoramidateanalog binding molecules are potentially powerful therapeutics foressentially any disease or condition that in some way involves DNA.Exemplary target sequences for such therapeutics include: a) DNAsequences involved in the propagation and/or maintenance infectiousagents, such as, bacterial, viruses, yeast and other fungi, for example,disrupting the metabolism of an infectious agent; and b) sequencescausing inappropriate expression or proliferation of cellular genes,such as oncogenes, for example, blocking or reducing the transcriptionof inappropriately expressed cellular genes (such as genes associatedwith certain genetic disorders).

Gene expression or replication can be blocked by generating triplexstructures in regions to which required regulatory proteins (ormolecules) are known to bind (for example, HIV transcription associatedfactors like promoter initiation sites and binding sites, McShan, etal.). Alternatively, specific sequences within protein-coding regions ofgenes (e.g., oncogenes) can be targeted as well.

When a phosphoramidate analog oligonucleotide is identified that binds aselected duplex target sequence tests, for example, by the gel bandshift mobility assay described above, the analog is further tested forits ability to form stable triplex structures in vitro. Cell culture andin vivo assay systems, such as those described above under "Anti-SenseApplications" are used.

Target sites can be chosen in the control region of the genes, e.g., inthe transcription initiation site or binding regions of regulatoryproteins (Helene and Toulme, 1990; Birg et al., 1990; Postel et al.,1991; Cooney et al., 1988). Also, target sites can be chosen such thatthe target also exists in mRNA sequences (i.e., a transcribed sequence),allowing oligonucleotides directed against the site to function asantisense mediators as well (see above).

Also, phosphoramidate modified DNA molecules can be used to generatetriplex molecules with a third strand target (i.e., a single-strandnucleic acid). For example, a DNA molecule having two regions capable offorming a triplex structure with a selected target third strand moleculecan be synthesized. Typically the two regions are linked by a flexibleregion which allows the association of the two regions with the thirdstrand to form a triplex. One example of such a DNA molecule is T₁₀(fully phosphoramidate modified)-C₄ (hinge region)-T₁₀ (phosphodiesterlinkages). This molecule forms triplex structures with a polyA RNAtarget. A corresponding DNA molecule having T₁₀ (phosphodiesterlinkages)-C₄ (hinge region)-T₁₀ (phosphodiester linkages) does not formtriplex with a polyA RNA target.

Hinge regions can comprise any flexible linkage that keeps the twotriplex forming regions together and allows them to associate with thethird strand to form the triplex. Third strand targets are selected tohave appropriate purine/pyrimidine content so as to allow formation oftriplex molecules.

The flexible linkage may connect the two triplex forming regions(typically, complementary DNA strands) in any selected orientationdepending on the nature of the base sequence of the target. For example,the two triplex forming regions each have 5' and 3' ends, these ends canbe connected by the flexible hinge region in the following orientations:5' to 3', 3' to 5', 3' to 3', and 5' to 5'.

Further, duplex DNA molecules containing at least one phosphoramidatelinkage in each strand can be used as decoy molecules for transcriptionfactors or DNA binding proteins (e.g., c-myb).

Single-stranded DNA can also be used as a target nucleic acid foroligonucleotides of the present invention, using, for example,phosphoramidate intersubunit linkage-containing hairpin structures(e.g., FIG. 6). Two phosphoramidate analog oligonucleotides can beselected for single-strand DNA target-directed binding. Binding of thetwo phosphoramidate analog strands to the single-strand DNA targetresults in formation of a triplex.

C. PHARMACEUTICAL COMPOSITIONS

The present invention includes pharmaceutical compositions useful inantisense and antigene therapies. The compositions comprise an effectiveamount of N3'→P5' phosphoramidate oligonucleotides in combination with apharmaceutically acceptable carrier. One or more N3'→P5' phosphoramidateoligonucleotides (having different base sequences) may be included inany given formulation.

The N3'→P5' phosphoramidate oligonucleotides, when employed intherapeutic applications, can be formulated neat or with the addition ofa pharmaceutical carrier. The pharmaceutical carrier may be solid orliquid. The formulation is then administered in a therapeuticallyeffective dose to a subject in need thereof.

Liquid carriers can be used in the preparation of solutions, emulsions,suspensions and pressurized compositions. The N3'→P5' phosphoramidateoligonucleotides are dissolved or suspended in a pharmaceuticallyacceptable liquid carrier such as water, an organic solvent, a mixtureof both, or pharmaceutically accepted oils or fats. The liquid carriercan contain other suitable pharmaceutical additives including, but notlimited to, the following: solubilizers, suspending agents, emulsifiers,buffers, thickening agents, colors, viscosity regulators, preservatives,stabilizers and osmolarity regulators. Suitable examples of liquidcarriers for parenteral administration of N3'→P5' phosphoramidateoligonucleotides preparations include water (partially containingadditives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric alcohols andpolyhydric alcohols, e.g., glycols) and their derivatives, and oils(e.g., fractionated coconut oil and arachis oil).

For parenteral administration of N3'→P5' phosphoramidateoligonucleotides the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile carriers are useful in sterileliquid form compositions for parenteral administration.

Sterile liquid pharmaceutical compositions, solutions or suspensions canbe utilized by, for example, intraperitoneal injection, subcutaneousinjection, intravenously, or topically. For example, antisenseoligonucleotides directed against retinal cytomegalovirus infection maybe administered topically by eyedrops. N3'→P5' phosphoramidateoligonucleotides can be also be administered intravascularly or via avascular stent impregnated with mycophenolic acid, for example, duringballoon catheterization to provide localized anti-restenosis effectsimmediately following injury.

The liquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent. Suchpressurized compositions may also be lipid encapsulated for delivery viainhalation. For administration by intranasal or intrabronchialinhalation or insufflation, N3'→P5' phosphoramidate oligonucleotides maybe formulated into an aqueous or partially aqueous solution, which canthen be utilized in the form of an aerosol, for example, for treatmentof infections of the lungs like Pneumocystis carnii.

N3'→P5' phosphoramidate oligonucleotides may be administered topicallyas a solution, cream, or lotion, by formulation with pharmaceuticallyacceptable vehicles containing the active compound. For example, for thetreatment of genital warts.

The N3'→P5' phosphoramidate oligonucleotides may be administered inliposome carriers. The use of liposomes to facilitate cellular uptake isdescribed, for example, in U.S. Pat. Nos. 4,897,355 (Eppstein, D., etal., issued 30 Jan. 1990) and 4,394,448 (Szoka, F,, et al., issued 19Jul. 1983). Numerous publications describe the formulation andpreparation of liposomes.

The dosage requirements for treatment with N3'→P5' phosphoramidateoligonucleotides vary with the particular compositions employed, theroute of administration, the severity of the symptoms presented, theform of N3'→P5' phosphoramidate oligonucleotides and the particularsubject being treated.

In general, N3'→P5' phosphoramidate oligonucleotides are administered ata concentration that affords effective results without causing anyharmful or deleterious side effects (e.g., an effective amount). Such aconcentration can be achieved by administration of either a single unitdose, or by the administration of the dose divided into convenientsubunits at suitable intervals throughout the day.

D. DIAGNOSTIC APPLICATIONS

The phosphoramidate analog oligonucleotides are also useful indiagnostic assays for detection of RNA or DNA having a given targetsequence. In one general application, the phosphoramidate analogs arelabeled (e.g., isotopically or other detectable reporter group) and usedas probes for DNA or RNA samples that bound to a solid support (e.g.,nylon membranes).

Alternatively, the phosphoramidate analog oligonucleotides may be boundto a solid support (for example, magnetic beads) and homologous RNA orDNA molecules in a sample separated from other components of the samplebased on their hybridization to the immobilized phosphoramidate analogs.Binding of phosphoramidate analogs to a solid support can be carried outby conventional methods. Presence of the bound RNA or DNA can bedetected by standard methods, for example, using a second labeledreporter or polymerase chain reaction (Mullis; Mullis, et al.).

Diagnostic assays can be carried out according to standard procedures,with suitable adjustment of the hybridization conditions to allowphosphoramidate analog hybridization to the target region. The abilityof phosphoramidate analog oligonucleotides to bind at elevatedtemperature can also help minimizes competition for binding to a targetsequence between the phosphoramidate analog probe and any correspondingsingle-strand phosphodiester oligonucleotide that is present in thediagnostic sample.

D. OTHER APPLICATIONS

In One aspect, the phosphoramidate analog oligonucleotides can be usedin methods to enhance isolation of RNA or DNA from samples. For example,as discussed above, phosphoramidate analogs can be fixed to a solidsupport and used to isolate complementary nucleic acid sequences, forexample, purification of a specific mRNA from a polyA fraction(Goldberg, et al). The phosphoramidate analogs are advantageous for suchapplications since they can form more stable interactions with RNA andduplex DNA than standard phosphodiester oligonucleotides. A large numberof applications in molecular biology can be found for reporter labeledphosphoramidate analogs, particularly for the detection of RNA insamples. Phosphoramidate analogs can be labeled with radioactivereporters (³ H, ¹⁴ C, ³² P, or ³⁵ S nucleosides), biotin or fluorescentlabels (Gryaznov, et al.). Labelled phosphoramidate analogoligonucleotides can be used as efficient probes in, for example, RNAhybridization reactions (Ausubel, et al., Sambrook, et al.).

Also, double-stranded DNA molecules where each strand contains at leastone phosphoramidate linkage can be used for the isolation of DNA-duplexbinding proteins. In this embodiment the duplex containingphosphoramidate intersubunit linkages is typically affixed to a solidsupport and sample containing a suspected binding protein is then passedover the support under buffer conditions that facilitate the binding ofthe protein to its DNA target. The protein is typically eluted from thecolumn by changing buffer conditions.

The triplex forming DNA molecules described above, containingphosphoramidate modified linkages, can be used as diagnostic reagents aswell, to, for example, detect the presence of an RNA molecule in asample.

Further, complexes containing oligodeoxyribonucleotides having N3'→P4'phosphoramidate intersubunit linkages can be used to screen for usefulsmall molecules or binding proteins: for example, N3'→P5'phosphoramidate oligodeoxyribonucleotide complexes with duplex DNA canbe used to screen for small molecules capable of further stabilizing thetriplex structure. Similar screens are useful with N3'→P5'phosphoramidate oligodeoxyribonucleotide complexes formed with singlestrand DNA and RNA molecules.

F. VARIATIONS

Variations on the phosphoramidate analog oligonucleotides used in themethods of the present invention include modifications to facilitateuptake of the oligonucleotide by the cell (e.g., the addition of acholesterol moiety (Letsinger, 1990); production of chimeticoligonucleotides using other intersubunit linkages (Goodchild);modification with intercalating agents (for example, triplex stabilizingintercalating agents, Wilson, et al., 1993); and use of ribose insteadof deoxyribose subunits.

Further modifications include, 5' and 3' terminal modifications to theoligonucleotides (e.g., --OH, --OR, --NHR, NH₂ and cholesterol). Inaddition, the ribose 2' position can be the site of numerousmodifications, including, but not limited to, halogenation (e.g., --F).

N3'→P5' phosphoramidate oligonucleotides may also be modified byconjugation to a polypeptide that is taken up by specific cells. Suchuseful polypeptides include peptide hormones, antigens and antibodies.For example, a polypeptide can be selected that is specifically taken upby a neoplastic cell, resulting in specific delivery of N3'→P5'phosphoramidate oligonucleotides to that cell type. The polypeptide andoligonucleotide can be coupled by means known in the art (see, forexample, PCT International Application Publication No. PCT/US89/02363,WO8912110, published 12/14/89, Ramachandr, K, et al.).

The properties of such modified phosphoramidate analog oligonucleotides,when applied to the methods of the present invention, can be determinedby the methods described herein.

While preferred embodiments, uses, and methods of practicing the presentinvention have been described in detail, it will be appreciated thatvarious other uses, formulations, and methods of practice as indicatedherein are within the contemplation of the present invention.

MATERIALS AND METHODS

The methyl and cyanoethyl phosphoramidates and the H-phosphatenucleoside reagents were purchased from Glen Research (Sterling, Va.)and Applied Biosystems Inc., (Foster City, Calif.) Nucleosidemethylphosphonoamidite reagents were purchased from Glen Research, andDMT-dT-LCAA CPG, 500Å, was purchased from Applied Biosystems Inc.

For enzymatic hydrolysis of oligonucleotides, 0.2 A₂₆₀ units of aselected oligonucleotide and 0.22 U of phosphodiesterase from Crotalusdurissus (Boehringer-Mannheim, Indianapolis, Ind.) were incubated for in100 μl 10 mM Tris-HCl and 10 mM MgCl₂. Samples were taken for analysisat 0', 10', 40', 4.5 hours and 22 hours after addition of thephosphodiesterase. Products were analyzed by RP HPLC essentially asdescribed in Example 2D.

Standard nucleic acid chemistry, including chemical synthesis of nucleicacids, has been reviewed by Miller (1990).

Chemicals were purchased from Aldrich (Milwaukee, Wis.), Sigma (St.Louis, Mo.) and Calbiochem (San Diego, Calif.).

HPLC was typically carried out using a Dionex chromatograph (Sunnyvale,Calif.). A "HYPERSIL ODS" column (4.6×200 mm, 5μ particle size; HewlettPackard, Palo Alto, Calif.) and a 0.5%/minute gradient of CH₃ CN in 0.05M TEAH buffer, pH 7.0, were used for RP HPLC. For ion exchangechromatography, a Dionex "OMNI PAK" NA 100 column (4×250 mm) was usedwith a 1%/minute gradient of 1.5 M NaCl in water. "NAP 5" columns fromPharmacia (Uppsala, Sweden) were used for desalting of oligonucleotides.Capillary electrophoresis (CE) analysis was performed on an ABI 270Asystem with 10% MICROGEL™ capillaries (0.1×500 mm) in 35 mM Tris-boratebuffer, pH 9.0. Thermal dissociation experiments were done on a VarianIE spectrophotometer and temperature controller. Absorbance values at260 nm or 284 nm were obtained at 1 minute intervals at a heating rateof 1.0° C./minute.

EXAMPLE 1 SYNTHESIS OF OLIGONUCLEOTIDES CONTAININGOLIGODEOXYRIBONUCLEOTIDE N3'→P5' PHOSPHORAMIDATES

A. GENERAL METHODS

Synthesis of the phosphoramidate analogs was carried out either manuallyin syringe or automatically on ABI 384 synthesizer (ABI, Foster City,Calif.).

FIG. 1E presents a schematic representation of the synthesis ofuniformly modified oligonucleotides on a solid support using astep-by-step elongation procedure.

For a given cycle the chemical steps, reagents, and reaction times were(i) detritylation, 3% dichloroacetic acid in dichloromethane, 1.5 min.(FIG. 1, step i); (ii) phosphitylation, 0.2 M2-cyanoethyl-N,N-diisopropylchlorophospine and 0.2 Mdiisopropylethylamine in dichloromethane, 10 min. (FIG. 1, steps ii);(iii) hydrolysis, 0.4 M tetrazole in acetonitrile/water, 9/1 v/v, 5 min.(FIG. 1, step iii); (iv) coupling, 0.2 M 5'-DMT-3' amino nucleoside and0.2 M triethylamine in carbon tetrachloride/acetonitrile, 1/1, v/v, 20min (FIG. 1, step iv).

Standard oligonucleotides have phosphodiester intersubunit linkages weresynthesized by standard methods (ABI 384 synthesizer).

To construct chimeric oligomers, 5'-DMT-N-protected 3'-phosphoramidatedimer building blocks, having a 3'-NHP(O)(OCE)O-5' phosphoramidateinternucleoside linkage group, were used for synthesis with theconventional phosphoramidate method (essentially as previously describedby Gryaznov, et al., herein incorporated by reference).

Exemplary oligonucleotides synthesized by the method of the presentinvention are presented in FIG. 3. Further details of synthesis followhere.

B. MANUAL SYNTHESIS OF THE OLIGONUCLEOTIDE N3'→P5' PHOSPHORAMIDATES

Controlled pore glass (CPG) polymer support containing 1 μmol of5'-DMT-N-protected nucleoside was placed in a 1 ml Hamilton gas tightsyringe equipped with a plug of glass wool at the base.

For a given cycle of synthesis, reagents were drawn in and expelled fromthe syringe according to the following protocol:

1. Detritylation--3% dichloroacetic acid in dichloromethane, 5×0.5 ml:1.5 min.

2. Washing--acetonitrile, 6×0.5 ml.

3. Phosphitilation--0.2 M 2-cyanoethyl-N,N-diisopropylchlorophospine and0.2 M diisopropylethylamine in dichloromethane, 0.5 ml, 10 min withperiodic shaking.

4. Hydrolysis--0.4 M tetrazole in acetonitrile/water, 9/1, v/v, 5minutes with periodic shaking.

5. Washing--anhydrous acetonitrile, 10×0.5ml.

6. Coupling--0.2 M 5'-DMT-3'amino nucleoside and 0.2 M triethylamine incarbon tetrachloride/acetonitrile, 1/1, v/v, 20 minutes, with shaking.After coupling, the solution was collected to recover un-reactednucleoside.

7. Washing--acetonitrile, 6×0.5 ml.

The steps 1-7 were repeated until the desired oligonucleotide wasprepared. The average coupling yields were 94%-96% as judged byDMT-cation assay. On completion of the cycles, the support-boundoligomer was detritylated. Cleavage from the support and N-deprotectionwith concentrated ammonium hydroxide afforded crude oligonucleotideswhich were purified by ion exchange HPLC.

C. MANUAL SYNTHESIS OF THE OLIGONUCLEOTIDE 2 CONTAINING ALTERNATIVEN3'→P5' PHOSPHORAMIDATE O3'→P5 ' PHOSPHODIESTER LINKAGES

5'-DMT-3'-amino thymidine and 5'-DMT-thymidine-3'-phosphoramidatesubunits were used to synthesize oligonucleotides having alternativeN3'→P5' phosphoramidate and O3'→P5' phosphodiester linkages.

For the synthesis of oligonucleotide 2 (SEQ ID NO:2), one micromole ofT-CPG (Thymidine-linked-CPG) was placed in a 1 ml Hamilton gas tightsyringe. Addition of 5'-DMT-3'-amino thymidine subunits was carried outas described above. Addition of 5'-DMT-thymidine-3'-phosphoramidatesubunits was carried out by standard synthetic procedures (AppliedBiosystems, Foster City Calif.).

After the 9th coupling reaction, the polymer support was treated withconcentrated ammonia hydroxide to release the crude oligomer. Theoligonucleotide was purified by ion exchange HPLC (e.g., Example 2).Oligonucleotide 2 was analyzed by CE and ³¹ P NMR (e.g., Example 2).

D. SYNTHESIS OF THE 5'-DMT-N-ISOBUTYRYL-3'-AMINO-2',3'-DIDEOXYGUANOSINE

The following steps describe a method of synthesis of5'-DMT-N-isobutyryl-3'-amino-2',3'-dideoxyguanosine.

1. 5 '-O-BENZOYL-N-ISOBUTYRYL-2'-DEOXYGUANOSINE was prepared fromN-isobutyryl-2'-deoxyguanosine essentially according to the method ofNishino, et al. (1986), except that the product was purified as follows:partitioning between CH₂ Cl₂ and water, concentrating the CH₂ Cl₂ layerin vacuo, and crystallizing the product with ether. After recovery byfiltration, the product was stirred overnight in fresh ether andrecollected by filtration. The overall yield of5'-O-benzoyl-N-isobutyryl-2'-deoxyguanosine was 50%-80%.

2. 5'-O-BENZOYL-N-ISOBUTYRYL-2'-DEOXYXYLOGUANOSINE was prepared from5'-O-benzoyl-N-isobutyryl-2'-deoxyguanosine essentially according to themethod of Herdewijn and van Aerschot (1989). The product was purified bydrying the crude mixture in vacuo, then dissolving in CH₂ Cl₂. The3'-O-benzoyl-N-isobutyryl-2'-deoxyxloguanosine product spontaneouslyprecipitated and was obtained by filtration.

3.5'-O-(4,4'-DIMETHOXYTRITYL)-3'-O-BENZOYL-N-ISOBUTYRYL-2'--DEOXYXYLOGUANOSINE.Dry 3'-O-benzoyl-N-isobutyryl-2'-deoxyxyloguanosine (7.3 g) and 7.9 g4,4'-dimethoxytrityl chloride were dissolved in 150 mL anhydrouspyridine. After 24 hours, 1 mL of water was added to the mixture. Themixture was then concentrated in vacuo to a foam containing the5'-O(4,4'-dimethoxytrityl)-3'-O-benzoyl-N-isobutyryl-2'-deoxyxyloguanosineproduct. The foam was dissolved in 300 mL CH₂ Cl₂, washed with 250 mLwater, and reconcentrated in vacuo.

4. 5'-O-(4,4'-DIMETHOXYTRITYL)-N-ISOBUTYRL-2'-DEOXYXYLOGUANOSINE. Thecrude5'-O-(4,4'-dimethoxytrityl)-3'-O-benzoyl-N-isobutyryl-2'-deoxyxyloguanosinewas dissolved in 1.2 L 5:4:1 dioxane:methanol:water and cooled in an icebath. To this mixture, 120 mL 2 N NaOH was added. The resulting mixturewas stirred at 0° C. for 25 minutes, and neutralized with pyridinium H⁺-form Dowex 50 ion exchange resin. After 2-3 minutes, the resin wasremoved by filtration and the product concentrated to a slurry in vacuo.The white precipitate in the slurry(5'-O-(4,4'-dimethoxytrityl)-N-isobutyryl-2'-deoxyxyloquanosine) wasremoved by filtration, washed with water, air dried and desiccated undervacuum over P₂ O₅.

5. 5 ' -O-(4,4 '-DIMETHOXYTRITYL)-3-AMINO-N-ISOBUTYRYL-2',3'-DIDEOXYGUANOSINE. To thecrude 5'-O-(4,4'-dimethoxytrityl)-N-isobutyryl-2'-deoxyxyloguanosine,7.9 g triphenylphosphine and 4.8 g LiN₃ were added. The mixture wasfurther dried under vacuum over P₂ O₅ for 3 hours. These dry compoundswere dissolved in 450 mL anhydrous dimethylformamide. To this solution5.3 mL diethyl azodicarboxylate was added. The mixture was stirredovernight, 1 mL water added, and the solvent removed in vacuo.

One liter CH₂ Cl₂ was added to the dried mixture and the resultingmixture washed twice with 1 L of water each time. The CH₂ Cl₂ layer wasconcentrated in vacuo to a light brown oil which was then dissolved in600 mL 10% triethylamine in pyridine. This mixture was cooled in an icebath and H₂ S added by bubbling. After 30 minutes, the ice bath wasremoved, and the H₂ S stream continued another 3 hours. The solution wasconcentrated in vacuo to a light brown oil.

Flash chromatography of the oil on a silica gel column, pretreated with0.5% pyridine in CH₂ Cl₂, then eluted with a gradient of 0%-5% methanolin CH₂ Cl₂ produced 3.5 g5'-O-(4,4'-dimethoxytrityl)-3-amino-N-isobutyryl-2',3'-dideoxyguanosine(FIG. 1C).

E. SYNTHESIS OF THE 5 '-DMT-N-BENZOYL-3'-AMINO-2',3'-DIDEOXYADENOSINE

The steps of the synthesis of 3'-amino-N⁶ -5'-dimethoxytrityl-2',3'-dideooxy-adenosine are illustrated in FIG. 20.

1. PREPARATION OF N⁶ 5'-DIBENZOYL-2'-DEOXYADENOSINE. A solution ofbenzoyl chloride (2.45 mL, 21 mmol) in pyridine (150 mL) was addeddrop-wise to a solution of N⁶ -benzoyl-2'-deoxyadenosine (compound 1,FIG. 20) (5 g, 14 mmol) in pyridine (45 mL) at room temperature overapproximately 1 hour. The reaction mixture was stirred at roomtemperature for an additional hour. The reaction mixture was quenchedwith methanol (5 mL) and evaporated to dryness.

The residue was dissolved in CH₂ Cl₂, washed with sat. aq. NaHCO₃ and H₂O. The organic layer was then dried over Na₂ S₄ and evaporated todryness. This residue was dissolved in CH₂ Cl₂ and subjected to columnchromatography (silica gel, 70-230 mesh, 200 g) , eluted with 5% CH₃OH/CH₂ Cl₂, and the solvent removed by evaporation, yielding 6 g (93%)of the desired N⁶ 5'-dibenzoyl-2'-deoxyadenosine product (compound 2,FIG. 20).

2. PREPARATION OF N⁶-BENZOYL-9-(3-O-BENZOYL-2-DEOXY-β-D-THREO-PENTEOFURANOSYL) ADENINE.Trifluoromethane sulfonic anhydride (4.2 mL, 25 mmol), was added to thesuspension of N⁶ 5'-dibenzoyl-2'-deoxyadenosine (7.5 g, 16.3 mmol) in10% pyridine/CH₂ Cl₂ (150 ML) at 0° C. The reaction was stirred at 0° C.for 30 minutes followed by the addition of H₂ O (26 mL). The reactionmixture was then stirred at room temperature overnight, and evaporatedto dryness.

The residue was redissolved in CH₂ Cl₂, washed with sat. aq. NaHCO₂, H₂O, and dried over Na₂ SO₄. The organic layer was evaporated to drynessto give brown oil, containing the N⁶-benzoyl-9-(3-O-benzoyl-2-deoxy-β-D-THREO-PENTEOFURANOSYL) ADENINEproduct (compound 3, FIG. 20), which was used without furtherpurification.

3. PREPARATION OF N⁶-BENZOYL-9-(3-O-BENZOYL-5-DIMETHOXYTRITYL-2-DEOXY-β-D-THREO-PENTOFURANOSYL)ADENINE. N⁶ -benzoyl-9-(3-O-benzoyl-2-deoxy-β-D-THREO-PENTEOFURANOSYL)ADENINE was dissolved in pyridine (60 mL). Dimethozytrityl chloride wasthen stirred at room temperature overnight. The reaction was quenchedwith CH₃ OH. The mixture was evaporated to dryness. The residue wasredissolved in CH₂ Cl₂, washed with sat. aq. NaHCO₃, H₂ O, dried overNa₂ SO₄ and evaporated. The residue was dissolved in CH₂ Cl₂ andsubjected to column chromatography (silica gel, 70-230 mesh, 400 g),eluted with 4% CH₃ OH/CH₂ Cl₂. The desired fraction containing the N⁶-benzoyl-9-(3-O-benzoyl-5-dimethoxytrityl-2-deoxy-β-D-THREO-PENTOFURANOSYL)ADENINE PRODUCT (COMPOUND 4, FIG. 20) was collected and evaporated todryness. The yield of product was 7 g (56% from N⁶5'-dibenzoyl-2'-deoxyadenosine).

4. PREPARATION OF N⁶ -BENZOYL-9-(5-O-DIMETHOXYTRITYL-2-DEOXY-β-D-THREO-PENTOFURANOSYL) ADENINE). Aqueous NaOH (2 M, 40 mL) wasadded to a solution of N⁶-benzoyl-9-(3-O-benzoyl-5-dimethoxytrityl-2-deoxy-β-D-THREO-PENTOFURANOSYL)ADENINE (7 g, 9.2 mmol) in a mixture of p-Dioxane (200 mL), CH₃ OH (160ML) and H₂ O (40 mL) at 0° C. The reaction mixture was stirred at 0° C.for 25 minutes. The reaction was terminated by the addition of Dowex50×2-100 ion-exchange resin (pyridinium form) to neutralize the solutionto pH 7. The solid was removed by filtration and the solvent evaporatedto dryness.

The residue was dissolved in CH₂ Cl₂, washed with sat. aq. NaHCO₃, driedover Na₂ SO₄ and evaporated to dryness to give light yellow solidcontaining the N⁶-benzoyl-9-(5-O-dimethoxytrityl-2-deoxy-β-D-THREO-PENTOFURANOSYL)ADENINE PRODUCT (COMPOUND 5, FIG. 20). This product was used withoutfurther purification. The yield of product from the previous step was 6g (99%).

5. PREPARATION OF 3'-AMINO-N⁶-BENZOYL-5'-DIMETHOXYTRITYL-2',3'-DIDEOXY-ADENOSINE.Diethylazodicarboxylate (1.42 mL, 9 mmol) was added to a suspension ofN⁶ -benzoyl-9-(5-O-dimethoxytrityl-2-deoxy-β-D-THREO-PENTOFURANOSYL)ADENINE) (2 g, 3 mmol) in DMF (40 mL), the suspension further containingtriphenylphosphine (2.4 g, 9 mmol) and LiN₃ (4.1 g, 83.8 mmol). Thereaction mixture was stirred at room temperature overnight andevaporated to dryness. The residue was dissolved in CH₂ Cl₂, washed withsat. aq. NaHCO₃, H₂ O, dried over Na₂ SO₄ and evaporated to give a brownoil. The brown oil was dissolved in CH₂ Cl₂ and subjected to columnchromatography (silica gel, 70-230 mesh, 100 g), eluted with 3% CH₃OH/CH₂ Cl₂ to give compound 6 (FIG. 20) as light brown oil.

This oil was dissolved in 15% triethylamine/pyridine (36 mL). Hydrogensulfide was then bubbled into the solution at 0° C. for 30 minutes. Thesolution was stirred at room temperature for 30 more minutes. Thesolvent was evaporated to dryness. The residue was dissolved in CH₂ Cl₂,washed with sat. aq. NaHCO₂, H₂ O, dried over Na₂ SO₄ and evaporated todryness. The residue was dissolved in CH₂ Cl₂ and subjected to columnchromatography (silica gel, 70-230 mesh, 100 g), eluted with 5% CH₃OH/CH₂ Cl₂ to give the final product 3'-amino-N⁶-5'-dimethoxytrityl-2',3'-dideooxy-adenosine (compound 7, FIG. 20; FIG.1). The yield of the product from the previous step was 1.6 g (80%).

E. SYNTHESIS OF OTHER DMT-SUBUNITS

Synthesis of the 5'-DMT-3'amino thymidine and 5'-DMT-N-benzoyl-3'-aminocytidine was performed according to Glinski, et al. (1970).

EXAMPLE 2 CHARACTERIZATION OF OLIGONUCLEOTIDES CONTAINING N3'→P5'PHOSPHORAMIDATE LINKAGES

Oligonucleotides synthesized as described in Example 1 were evaluated bythe following methods.

A. PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY

Oligonucleotides were purified away from excess reaction components byion exchange (IE) HPLC. IE HPLC analyses were performed on a Dionex(Sunnyvale, Calif.) chromatograph. A Dionex "OMNIPAC NA100," 4×250 mmcolumn was used, with a 1%/min or 2%/min gradient of 1.0 M NaCl in 0.03M TEAA buffer, pH 7.0; flow rate, 1.0 ml/min.

FIG. 4A presents an exemplary HPLC chromatogram of the reaction mixtureafter synthesis of the phosphoramidate Oligonucleotide 3 (FIG. 3). Thelargest peak in the figure corresponds to the Oligonucleotide 3 product.Retention time of the 3'-NHP(O)(O⁻)O-5' phosphoramidates on IE HPLCcolumn was 1.0-1.5 minutes shorter than the retention time forcorresponding phosphodiester compounds. The product was thenconcentrated by precipitation with ethanol and resuspended in water.

B. PURITY OF OLIGONUCLEOTIDES

Purity of synthesized oligonucleotides was typically evaluated bycapillary gel electrophoresis. Capillary electrophoresis was performedusing an Applied Biosystems Incorporated Model 270A machine, on"MICKROGEL" capillary tubes, essentially following the directions of themanufacturer.

FIG. 4B presents an exemplary capillary gel electrophoresis profile ofthe reaction mixture after synthesis of the undecaphosphoramidate 6(FIG. 3).

Alternatively, purity of the isolated oligonucleotides is evaluated byelectrophoretic separation of the samples in high percent polyacrylamidegels, for example, 20% acrylamide, 5% bis-acrylamide (Ausubel, et al.;Maniatis, et al.). Oligonucleotides were visualized by staining withethidium bromide and exposure to UV light. Relative mobilities inpolyacrylamide gels upon electrophoretic separation of the3'-NHP(O)(O⁻)O-5' phosphoramidates was 10%-15% lower than for thecorresponding phosphodiester compounds.

C. NUCLEAR MAGNETIC RESONANCE ANALYSIS

NMR spectra were recorded on a Varian XL-400 (Varian Associates, PaloAlto, Calif.) spectrometer at 162 MHz for ³¹ P spectra, with 85%phosphoric acid in D₂ O as an external standard, and at 400 MHz for ¹ Hspectra, with tetramethyl silane (TMS) as an external standard.

Exemplary results are presented in FIG. 4C, showing the ³¹ P-NMRspectrum of the purified decaphosphoramidate oligonucleotide 3 (FIG. 3).The spectrum presents a peak at δ, ppm, 7.12 which is characteristic ofphosphoramidate groups.

D. REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OFHYDROLYSIS PRODUCTS

Purified phosphoramidate 10-mer oligonucleotide 3 (FIG. 3) washydrolysed by treatment with 80% acetic acid for 48 hours at 25° C. Thehydrolysis products were evaluated by reverse phased (RP) PIPLC. RP HPLCanalyses were performed with a Dionex chromatograph on a "HYPERSIL ODS"5μ, 4.6×200 mm column from Hewlett Packard (Palo Alto, Calif.), using a1% min gradient of acetonitrile in 0.03 M TEAA buffer, pH 7.0; flowrate, 1 ml/min. The hydrolysis products were identified as 3'amino-5'-thymidilic acid, 5'-thymidilic acid, and 3'-aminothymidine.Further, about 7% of the hydrolysis products (total peaks) were minorby-products. These results confirm the presence of N3'→P5'phosphoramidate linkages in the oligonucleotide.

EXAMPLE 3 THERMAL DISSOCIATION

A. DUPLEX MELTING

Thermal dissociation curves were obtained using a Carry 1Espectrophotometer (Varian, Palo Alto, Calif.) that was equipped with atemperature controller (Varian). The reaction solutions containedequivalent concentrations of oligomer and complement (approximately 6 μMin oligomer strand) in 15 mM phosphate buffer at pH 7.05, with NaCladded to give a total Na+ concentration of 100 mM.

The molar extinction coefficients used for oligo(dT), poly(dA) andpoly(A) were 8.2, 8.4, and 10.2 A₂₆₀ ×10³, respectively. An extinctioncoefficient of 109 A₂₆₀ Units/μM was used for the mixed-basedoligomers--the extinction coefficient was calculated from the tablecompiled by P. N. Borer (1975). Reaction solutions were equilibrated at0° C. and the absorbance at 260 nm was followed as the temperature wasincreased in increments of 3° C. per 5 minutes. The fraction of anoligomer in the bound state, α, at a given temperature was determined byuse of upper and lower base lines as described by Abergo, et al. (1981).T_(m) values are defined as the temperature at which α=0.5. Plots of Ink(Marky, et al., 1987) versus 1/T were linear for these compounds.

The results of the thermal denaturation studies were also plotted asnormalized absorbance at 260 nm versus temperature in °C. FIGS. 5A and5B display exemplary melting curves for the duplexes formed byphosphodiester and phosphoramidate oligomers. In the figures: (A), (C)and (B), (D), respectively, correspond to FIG. 3, experiments 8, 9,using phosphodiester Oligonucleotide SEQ ID NO:4); and, FIG. 3,experiments 13, 14 using phosphoramidate Oligonucleotide SEQ ID NO:6).

The thermal stability data is summarized in FIG. 3 {T_(m) (°C.)}. In thetable, T_(m) is the temperature at the midpoint of the melting curve; npis the abbreviation for the 3'-NHP(O)(O⁻)O-5' phosphoramidate link.

The concentrations of oligonucleotides were typically at 5 μM oligomerstrands. Buffer A (10 mM Tris HCl, 150 mM NaCl, pH 7.02) was used forduplex thermal stability studies.

B. OLIGONUCLEOTIDE HAIRPINS

The stability of duplexes formed by oligonucleotides containingphosphoramidate linkages in both complementary strands was evaluatedessentially as described above. The chimericphosphoramidate-phosphodiester hairpin oligomers presented in FIG. 6were synthesized.

All the molecules in FIG. 6 were constructed using2'-deoxyribonucleotides.

Thermal dissociation experiments were performed essentially as describedabove, but with the following reaction conditions: 10 mM Tris HClbuffer, pH 7.02 at oligonucleotide concentration 2.5 μM.

The T_(m) values obtained from these experiments are summarized in FIG.6.

C. TRIPLEX MELTING

Triplex thermal stabilities were evaluated essentially as describedabove for duplexes. Buffer conditions were either as described above(Buffer A) or a second buffer, Buffer B (10 mM Tris HCl, 150 mM NaCl, 10mM MgCl₂, pH 7.02.) was used.

FIGS. 7A to 7D present exemplary triplex melting curves. FIGS. 7A and 7Cpresent normalized absorbance at 260 nm plotted against temperature.FIGS. 7B and 7D present normalized absorbance at 284 nm plotted againsttemperature. In the figures: the triplexes formed by double stranded DNAand phosphoramidate Oligonucleotide SEQ ID NO:3 correspond to the blackcircle line; triplexes formed with phosphodiester Oligonucleotide SEQ IDNO:1 correspond to open squares line).

The data correspond to FIG. 3, experiment 7 where curves (A) and (C)were thermal stability studies performed using buffers A and B,respectively, with hyperchromicity monitored at 260 nm. Curves (B) and(D) are (A) and (C) respectively, but where hyperchromicity wasmonitored at 284 nm.

The data presented in FIGS. 7A to 7D indicate that phosphodiesterOligonucleotide SEQ ID NO:1 did not form triplexes with the samedouble-stranded DNA targets as did phosphoramidate Oligonucleotide SEQID NO:3.

EXAMPLE 4 GEL BAND MOBILITY SHIFT ASSAYS

Triplex structures were further evaluated using gel band mobility shiftassays.

A. PHOSPHORAMIDATE OLIGONUCLEOTIDE SEQ ID NO:3

Gel band mobility shift assay conditions were as follows: 20%acrylamide, 5% bis-acrylamide in 10 mM MgCl₂, 80 mM Tris-borate buffer,pH 8.2, 10° C. Gels were typically run under native (non-denaturing)conditions. Exemplary results of such a gel band mobility shift assayare presented in FIG. 8. In the figure the lanes are as follows:1-10-mer phosphodiester Oligonucleotide SEQ ID NO:1; 2-10-merphosphoramidate Oligonucleotide SEQ ID NO:3; 3-24-mer hairpin targetd(A₁₀ C₄ T₁₀) (FIG. 3, experiment 7). In lane 3 the slow moving minorband likely corresponds to a bi-molecular duplex of d(A₁₀ C₄ T₁₀);4--hairpin target and Oligonucleotide SEQ ID NO:1; 5--hairpin target andOligonucleotide SEQ ID NO:3.

The gel was stained with STAINS-ALL™ (Kodak, Rochester N.Y.) and imagedon a Molecular Dynamics (Sunnyvale, Calif.) densitometer. The efficiencyof the phosphoramidate Oligonucleotide SEQ ID NO:3 staining is differentfrom phosphodiester compounds (Oligonucleotide SEQ ID NO:1 and theduplex).

In the figure the mobility of the triplex structure is denoted by arrow.As can be seen from the results presented in the figure, the gel bandmobility shift assay results confirm the results obtained from thermaldenaturation studies, i.e., that Oligonucleotide SEQ ID NO:1 fails toform a triplex with the same target as Oligonucleotide SEQ ID NO:3.

B. PHOSPHORAMIDATE OLIGONUCLEOTIDE SEQ ID NO:6

The triplex formation of phosphoramidate Oligonucleotide SEQ ID NO:6analog with a double-stranded DNA target was also confirmed by gel bandmobility shift assay.

The conditions of the gel band mobility shift assay were essentially asdescribed above. The results of the analyses are presented in FIG. 9. Inthe figure the lanes were as follows: 1-11-mer phosphodiesterOligonucleotide SEQ ID NO:4; 2-11-mer phosphoramidate OligonucleotideSEQ ID NO:6; 3-26-mer hairpin target (SEQ ID NO:22); 4--hairpin targetand Oligonucleotide SEQ ID NO:4; 5--hairpin target and OligonucleotideSEQ ID NO:6.

In the figure the mobility of the triplex structure is denoted by arrow.As seen above, the phosphodiester Oligonucleotide SEQ ID NO:4 fails toform a triplex with the same target as Oligonucleotide SEQ ID NO:6.

EXAMPLE 5 IN VITRO EVALUATION OF PHOSPHORAMIDATE ANALOGS

N3'→P5' phosphoramidate linkage containing anti-sense oligonucleotideswere synthesized which were complementary to the BCR/ABL fusion junction(B2A2) in leukemic cell line BV173. The following assay measuresleukemic cell proliferation and is performed essentially as described byAnfossi, et al. (1989).

Briefly, FIG. 10 illustrates the effect of BCR-ABL oligomers on leukemiccell proliferation of BV173 cells carrying the B2A2-type break points.BV173 is a leukemic cell line with the B2A2 BCR/ABL fusion junction(Pegoraro, et al., 1983). HL60 is a promyelocytic leukemic cell linewith a normal c-abl locus (Collins, et al., 1977). K562 is a leukemiccell line with the B3A2 BCR/ABL fusion junction (Seelig, et al., 1993).

Cells (5×10⁴) were placed in 0.2 ml of liquid suspension culture(Iscove's modified Dulbecco's modified medium with 2% human ABserum--Life Technologies, Gaithersburg, Md.).

The oligonucleotides were administered to the cultures at 24 hourintervals for three days (days 0, 1 and 2) to achieve the incrementalconcentrations shown in the legends of FIGS. 10-19 (all concentrationsin μg/mL), for example, FIG. 10--40/20/20; 20/10/10; 10/5/5; and5/2.5/2.5. Note that, for example, 40/20/20 reflects a final totalconcentration of 80 μg/ml in the cell culture medium after the lastoligonucleotide addition. Cell counts were performed by standardmethods.

FIG. 10 shows the results of the assay using fully modified N3'→P5'phosphoramidate oligonucleotide 6 (SEQ ID NO:6) at the concentrationsshown in the figure legend. The target cells were BV173 cells. Theresults demonstrate that the phosphoramidate oligonucleotide isextremely effective at inhibiting the growth of the leukemia cells--evenat the lowest concentration used.

FIG. 11 shows the results of treatment of the HL60 cells, a non-BRC-ABLexpressing cell line, with oligonucleotide SEQ ID NO:6. The resultsdemonstrate that at all concentrations the oligonucleotide was welltolerated by the cells. There was no apparent cytotoxicity.

FIGS. 12 and 13 show the results of similar experiments to the resultspresented in FIGS. 10 and 11, respectively. Even at the lowestconcentrations (0.3125/0.15625/0.15625) the N3'→P5' phosphoramidateoligonucleotide SEQ ID NO:6 is extremely effective at inhibitingleukemia cell proliferation (FIG. 12) while maintaining negligibletoxicity (FIG. 13).

FIGS. 14 and 15 show the results of similar experiments to the resultspresented in FIGS. 10 and 11, respectively. However, in the experimentcorresponding to the data presented in FIG. 15 the target cell was K562,which contains a B3A2 BCR/ABL fusion junction. In the data from theexperiment presented in FIG. 14, transition concentrations for theeffectiveness of the N3'→P5' phosphoramidate oligonucleotide SEQ ID NO:6in culture are shown. Specifically, at a concentration of0.0198/0.0098/0.0098 some release of inhibition of proliferation can beseen with oligonucleotide SEQ ID NO:6.

Further, the data presented in FIG. 15 demonstrate that phosphoramidateoligonucleotide SEQ ID NO:6 had essentially no effect on theproliferation of K562 cells, having the B3A2 fusion Junction. Theseresults demonstrate that the antiproliferation effect is specificallyassociated with the B2A2 BCR/ABL fusion junction to whichphosphoramidate oligonucleotide SEQ ID NO:6 is complementary.

In comparison, FIGS. 16-19 present similar data for a 16-mer (SEQ IDNO:26) having fully modified phosphorothioate intersubunit linkages.

FIG. 16 shows the results of the assay using fully modifiedphosphorothioate 16-mer (SEQ ID NO:26) at the concentrations shown inthe figure legend where the target cell line was BV173. The results aresimilar to those seen with oligonucleotide SEQ ID NO:6 (FIG. 10), butthe data demonstrate that the phosphorothioate oligonucleotide is not aseffective as oligonucleotide SEQ ID NO:6 at inhibiting the growth of theleukemia cells.

FIG. 17 shows the results of treatment of the BV173 BRC/ABL cell linewith a 16-met phosphorothioate oligonucleotide (SEQ ID NO:27) having asequence complementary to the B3A2 BCR/ABL fusion junction: B3A2 has a 2base pair sequence mismatch to the B2A2 BCR/ABL fusion junction. Theresults demonstrate that at all concentrations the oligonucleotide wastolerated by the cells.

FIGS. 18 and 19 show the results of similar experiments to the resultspresented in FIGS. 16 and 17, respectively. In the data from theseexperiments, the release of the inhibition of cellular proliferation isseen at much lower concentrations for the phosphorothioateoligonucleotide than was seen for the N3'→P5' phosphoramidateoligonucleotide SEQ ID NO:6.

These results demonstrate the in vitro N3'→P5' phosphoramidateoligonucleotides are effective antisense compounds at lowconcentrations. Further, the N3'→P5' phosphoramidate oligonucleotidesare better antisense agents than phosphorothioate oligonucleotides.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 27                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 1, Fig. 2                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTTTTTTTTT10                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 2, Fig. 2                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 5..6                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 7..8                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9..10                                                           (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TTTTTTTTTT10                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 3, Fig. 2                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: /note= "where the intersubunit                         bonds are "np""                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TTTTTTTTTTT11                                                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 4, Fig. 2                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTTCTTCCTTA11                                                                 (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RNA Oligonucleotide 5, Fig. 2                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTTCTTCCTTA11                                                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 6, Fig. 2                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: /note= "where the intersubunit                         bonds are "np""                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CTTCTTCCTTA11                                                                 (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 7, Fig. 5                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TATATATATATTTTTATATATATA24                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 8, Fig. 5                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 5..6                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 7..8                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TATATATATTTTTATATATA20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 9, Fig. 5                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 5..6                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 7..8                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9..10                                                           (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 15..16                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 17..18                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 19..20                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 21..22                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 23..24                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TATATATATATTTTTATATATATA24                                                    (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 10, Fig. 5                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TACGTACGTATTTTTACGTACGAT24                                                    (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 11, Fig. 5                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 5..6                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 7..8                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9..10                                                           (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TACGTACGTATTTTTACGTACGTA24                                                    (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Oligonucleotide 12, Fig. 5                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 5..6                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 7..8                                                            (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9..10                                                           (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 15..16                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 17..18                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 19..20                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 21..22                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 23..24                                                          (D) OTHER INFORMATION: /note= "where the intersubunit bond                    is "np""                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TACGTACGTATTTTTACGTACGTA24                                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Target, Experiment 7, Fig. 2                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AAAAAAAAAACCCCTTTTTTTTTT24                                                    (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Target, Experiment 8, Fig. 2                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ATAAGGAAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RNA Target, Experiment 9, Fig. 2                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AUAAGGAAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RNA Target, Experiment 10, Fig. 2                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AUAAGGUAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RNA Target, Experiment 11, Fig. 2                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AUAAGGAAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RNA Target, Experiment 12, Fig. 2                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AUAAGGUAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Target, Experiment 13, Fig. 2                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ATAAGGAAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RNA Target, Experiment 14, Fig. 2                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AUAAGGAAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: RNA Target, Experiment 15, Fig. 2                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AUAAGGUAGAAGC13                                                               (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: DNA Target Duplex, Fig. 8                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TTCCTTCTTTCTTTTGAAAGAAGGAA26                                                  (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 244 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: HIV-1 REV RESPONSE ELEMENT                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CAGUGGGAAUAGGAGCUUUGUUCCUUGGGUUCUUGGGAGCAGCAGGAAGCACUAUGGGCG60                CAGCGUCAAUGACGCUGACGGUACAGGCCAGACAAUUAUUGUCUGGUAUAGUGCAGCAGC120               AGAACAAUUUGCUGAGGGCUAUUGAGGCGCAACAGCAUCUGUUGCAACUCACAGUCUGGG180               GCAUCAAGCAGCUCCAGGCAAGAAUCCUGGCUGUGGAAAGAUACCUAAAGGAUCAACAGC240               UCCU244                                                                       (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: STEM II OF THE HIV-1 REV RESPONSE                     ELEMENT                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAGACAAUUAUUGUCUGGU60                AUAGUGCAG69                                                                   (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: the sequence of the TAR site of                       HIV-1                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGUCUCUCUGGUUAGACCAGAUCUGAGCCUGGGAGCUCUCUGGCUAACUAGAGAACCC58                  (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 16-mer, phosphorothioate                              intersubunit linkages                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AAGGGCTTCTTCCTTA16                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: 16-mer, phosphorothioate                              intersubunit linkages                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GAGTTCAAAAGCCCTT16                                                            __________________________________________________________________________

It is claimed:
 1. A method of hybridizing an oligodeoxyribonucleotide toan RNA target, comprising:(a) forming an oligodeoxyribonucleotidecomprising a sequence of nucleoside subunits joined by inter-subunitlinkages, wherein:(i) at least 50% of the inter-subunit linkages of saidoligodeoxyribonucleotide are N3'→P5' phosphoramidate inter-subunitlinkages of the formula:

    3'-NH-P(═O)(-O.sup.-)-O-5'; and

(ii) said oligodeoxyribonucleotide has at least 11 nucleotides; and (b)contacting the oligodeoxyribonucleotide with said RNA target to allowformation of a hybridization complex between saidoligodeoxyribonucleotide and said RNA target.
 2. The method of claim 1,wherein all of said inter-subunit linkages of saidoligodeoxyribonucleotide are N3'→N5' phosphoramidate linkages.
 3. Themethod of claim 1, wherein said inter-subunit linkages alternate N3'→P5'phosphoramidate linkage and a phosphodiester inter-subunit linkage. 4.The method of claim 1, wherein said oligodeoxyribonucleotide has 11 to24 nucleosides.
 5. The method of claim 1, wherein all of saidinter-subunit linkages are N3'→P5' phosphoramidate linkages.
 6. Themethod of claim 5, wherein said oligodeoxyribonucleotide has 11 to 50nucleosides.
 7. A kit for the isolation of a target RNA from a sample,the kit comprising: an oligodeoxyribonucleotide comprising a sequence ofnucleoside subunits joined by inter-subunit linkages wherein at least50% of the inter-subunit linkages of said oligodeoxyribonueleotide areN3'→P5' phosphoramidate inter-subunit linkages of the formula:

    3'-NH-P(═O)(-O.sup.-)-O-5'

wherein said oligodeoxyribonucleotide hybridizes to said target RNA, andsaid oligodeoxyribonucleotide has at least 11 nucleosides.
 8. The kit ofclaim 7, wherein all of said inter-subunit linkages of saidoligodeoxyribonucleotide are N3'→P5' phosphoramidate inter-subunitlinkages.
 9. The kit of claim 7, wherein said inter-subunit linkagesalternate N3'→P5' phosphoramidate inter-subunit linkages and aphosphodiester inter-subunit linkage.
 10. The kit of claim 7, whereinsaid oligodeoxyribonucleotide has 11 to 24 nucleosides.
 11. The kit ofclaim 7 further including a solid support attached to saidoligodeoxyribonucleotide.
 12. A diagnostic method to detect in a samplethe presence of an RNA having a selected target sequence comprising:(a)contacting an oligodeoxyribonucleotide with said RNA to allow formationof a hybridization complex between said oligodeoxyribonucleotide andsaid selected target sequence, the oligodeoxyribonucleotide comprising adefined sequence of nucleoside subunits and inter-subunit linkageswherein:(i) at least 50% of the inter-subunit linkages or saidoligodeoxyribonucleotide are N3'→P5' phosphoramidate inter-subunitlinkages of the formula:

    3'-NH-P(═O)(-O.sup.-)-O-5',

and (ii) said oligodeoxyribonucleotide has at least 11 nucleosides: and(b) detecting the presence of said hybridization complex, therebydetecting the presence of said RNA.
 13. The method of claim 12, whereinsaid oligodeoxyribonucleotide carries a reporter moiety and saiddetecting includes detection of said reporter moiety.
 14. The method ofclaim 13, wherein said reporter moiety is selected from the groupconsisting of radioactive labels, biotin labels, and fluorescent labels.15. The method of claim 12, wherein all of said inter-subunit linkagesof said oligodeoxyribonucleotide are N3'→P5' phosphoramidate linkages.16. The method of claim 12 wherein said inter-subunit linkages alternateN3'→P5' and a phosphodiester inter-subunit linkage.
 17. The method ofclaim 12, wherein said oligodeoxyribonucleotide has 11 to 24nucleotides.
 18. A method of hybridizing an oligodeoxyribonucleotide toan DNA target, comprising:(a) forming an oligodeoxyribonucleotidecomprising a sequence of nucleoside subunits joined by inter-subunitlinkages, wherein:(i) 100% of the inter-subunit linkages of saidoligodeoxyribonucleotide are N3'→P5' phosphoramidate inter-subunitlinkages of the formula:

    3'-NH-P(═O)(-O.sup.-)-O-5'; and

(ii) said oligodeoxyribonucleotide has at least 11 nucleotides; and (b)contacting the oligodeoxyribonucleotide with said DNA target to allowformation of a hybridization complex between saidoligodeoxyribonucleotide and said DNA target.
 19. The method of claim18, wherein said oligodeoxyribonucleotide carries a reporter moiety andsaid detecting includes detection of said reporter moiety.
 20. Themethod of claim 19, wherein said reporter moiety is selected from thegroup consisting of radioactive labels, biotin labels, and fluorescentlabels.
 21. The method of claim 18, wherein saidoligodeoxyribonucleotide has 11 to 24 nucleotides.
 22. The method ofclaim 18, wherein said oligodeoxyribonucleotide has 11 to 50nucleosides.
 23. The method of claim 18, wherein said DNA target issingle-stranded DNA.
 24. A diagnostic method to detect the presence of asingle-stranded DNA having a selected target sequence in a sample,comprising:(a) contacting an oligodeoxyribonucleotide with saidsingle-stranded DNA to allow formation of a hybridization complexbetween said oligodeoxyribonucleotide and said selected target sequence,the oligodeoxyribonucleotide comprising a defined sequence of nucleosidesubunits and inter-subunit linkages wherein:(i) 100% of theinter-subunit linkages of said oligodeoxyribonucleotide are N3'→P5'phosphoramidate inter-subunit linkages of the formula:

    3'-NH-P(═O)(-O.sup.-)-O-5',

and (ii) said oligodeoxyribonucleotide has at least 11 nucleosides; and(b) detecting the presence of said hybridization complex, therebydetecting the presence of said DNA.
 25. The method of claim 24, whereinsaid oligodeoxyribonucleotide carries a reporter moiety and saiddetecting includes detection of said reporter moiety.
 26. The method ofclaim 25, wherein said reporter moiety is selected from the groupconsisting of radioactive labels, biotin labels, and fluorescent labels.27. The method of claim 24, wherein said oligodeoxyribonucleotide has 11to 24 nucleosides.
 28. The method of claim 24, wherein saidoligodeoxyribonucleotide has 11 to 50 nucleosides.
 29. A method ofhybridizing an oligodeoxyribonucleotide to a polynucleotide targetsequence, the method comprising:forming an oligodeoxyribonucleotidecomprising a defined sequence of nucleoside subunits joined byinter-subunit linkages defined by the formula: ##STR1## wherein B is apurine or pyrimidine or an analog thereof and n is between 2 and 48; and(b) contacting the oligodeoxyribonucleotide with said polynucleotidetarget sequence to allow formation of a hybridization complex betweensaid oligodeoxyribonucleotide and said polynucleotide target sequence.30. The method of claim 29, wherein B of said oligodeoxyribonucleotideis selected from the group consisting of uracil, thymine, adenine,guanine, cytosine, 5-methylcytosine, 5-bromouracil, and inosine.
 31. Themethod of claim 30, wherein B of said oligodeoxyribonucleotide isselected from the group consisting of uracil, thymine, adenine, guanine,and cytosine.
 32. A kit for the isolation of a target polynucleotidefrom a sample, comprising:an oligodeoxyribonucleotide comprising adefined sequence of nucleoside subunits joined by inter-subunit linkagesand defined by the formula: ##STR2## wherein B is a purine or pyrimidineor an analog thereof and n is between 2 and 48 and wherein saidoligodeoxyribonucleotide hybridizes to said target polynucleotide. 33.The kit of claim 32, wherein B of said oligodeoxyribonucleotide isselected from the group consisting of uracil, thymine, adenine, guanine,cytosine, 5-methylcytosine, 5-bromouracil, and inosine.
 34. The kit ofclaim 32, wherein B of said oligodeoxyribonucleotide is selected fromthe group consisting of uracil, thymine, adenine, guanine, and cytosine.35. The kit of claim 32 further including a solid support attached tosaid oligodeoxyribonucleotide.
 36. A diagnostic method to detect thepresence of a polynucleotide having a selected target sequence in asample comprising:(a) contacting an oligodeoxyribonucleotide with saidpolynucleotide to allow formation of a hybridization complex betweensaid oligodeoxyribonucleotide and said target sequence, wherein saidoligodeoxyribonucleotide comprises a defined sequence of nucleosidesubunits joined by inter-subunit linkages defined by the formula:##STR3## wherein B is a purine or pyrimidine or an analog thereof and nis between 2 and 48; and (b) detecting the presence of saidhybridization complex, thereby detecting said polynucleotide.
 37. Themethod of claim 36, wherein B of said oligodeoxyribonucleotide isselected from the group consisting of uracil, thymine, adenine, guanine,cytosine, 5-methylcytosine, 5-bromouracil, and inosine.
 38. The methodof claim 37, wherein B of said oligodeoxyribonucleotide is selected fromthe group consisting of uracil, thymine, adenine, guanine, and cytosine.39. The method of claim 36, wherein said oligodeoxyribonucleotidecarries a reporter moiety and said detecting includes detection of saidreporter moiety.
 40. The method of claim 39, wherein said reportermoiety is selected from the group consisting of radioactive labels,biotin labels, and fluorescent labels.